PI: Domashevskiy, Artem Title: Development of a Novel Inhibitor of Ricin: A Potential Therapeutic Lead 

against Deadly Shiga and Related Toxins

Received: 06/22/2016 FOA: PA16-200 Council: 01/2017

Competition ID: FORMS-D FOA Title: Academic Research Enhancement Award (Parent R15)

1 R15 AI124044-01A1 Dual: Accession Number: 3950045

IPF: 1605024 Organization: JOHN JAY COLLEGE OF CRIMINAL JUSTICE

Former Number: Department: Sciences

IRG/SRG: MSFB AIDS: N Expedited: N

Subtotal Direct Costs 

(excludes consortium F&A) 

Year 1:

Animals: N 

Humans: N 

Clinical Trial: N

Current HS Code: 10

HESC: N

New Investigator: N

Early Stage Investigator: N

Senior/Key Personnel: Organization: Role Category:

Artem Domashevskiy Ph.D Research Foundation of CUNY o/b/o 

John Jay College

PD/PI 

Always follow your funding opportunity's instructions for application format. Although this application

demonstrates good grantsmanship, time has passed since the grantee applied. The samples may not 

reflect the latest format or rules. NIAID posts new samples periodically:

https://www.niaid.nih.gov/grants-contracts/sample-applications

The text of the application is copyrighted. The awardee provided express permission for NIAID to post

this grant application and summary statement for educational purposes. The awardee allows you to use

the material (e.g., data, writing, graphics) they shared in the applications for nonprofit educational 

purposes only, provided the material remains unchanged and the principal investigators, awardee 

organizations, and NIH NIAID are credited.

Freedom of Information Act (FOIA). NIAID is strongly committed to protecting the integrity and

confidentiality of the peer review process. When NIH responds to FOIA requests for grant applications and

summary statements, the material will be subject to FOIA exemptions and include substantial redactions. 

NIH must protect all confidential commercial or financial information, reviewer comments and

deliberations, and personal privacy information.

Note on Section 508 Conformance and Accessibility. We have reformatted this sample to improve 

accessibility for people with disabilities and users of assistive technology. If you have trouble accessing the

content, contact the NIAID Office of Knowledge and Educational Resources at deaweb@niaid.nih.gov.
OMB Number: 4040-0001

Expiration Date: 06/30/2016

Tracking Number: GRANT12199590 Funding Opportunity Number: PA-16-200 . Received Date:

2016-06-22T16:33:35.000-04:00

APPLICATION FOR FEDERAL ASSISTANCE

SF 424 (R&R)

3. DATE RECEIVED BY STATE State Application Identifier

1. TYPE OF SUBMISSION* 4.a. Federal Identifier

❍ Pre-application ❍ Application ● Changed/Corrected

Application

b. Agency Routing Number

2. DATE SUBMITTED Application Identifier c. Previous Grants.gov Tracking Number

GRANT12199564

5. APPLICANT INFORMATION Organizational DUNS*: 6201288630000

Legal Name*: Research Foundation of CUNY o/b/o John Jay College

Department:

Division:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

ZIP / Postal Code*:

Person to be contacted on matters involving this application

Prefix: First Name*: Artem Middle Name: Last Name*: Domashevskiy Suffix: Ph.D

Position/Title: Assistant Professor

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

ZIP / Postal Code*:

Phone Number*: Fax Number: Email: 

6. EMPLOYER IDENTIFICATION NUMBER (EIN) or (TIN)*

7. TYPE OF APPLICANT* H: Public/State Controlled Institution of Higher Education

Other (Specify):

Small Business Organization Type ❍ Women Owned ❍ Socially and Economically Disadvantaged

8. TYPE OF APPLICATION* If Revision, mark appropriate box(es).

❍ New ● Resubmission ❍ A. Increase Award ❍ B. Decrease Award ❍ C. Increase Duration

❍ Renewal ❍ Continuation ❍ Revision ❍ D. Decrease Duration ❍ E. Other (specify) :

Is this application being submitted to other agencies?* ❍Yes ●No What other Agencies?

9. NAME OF FEDERAL AGENCY*

National Institutes of Health

10. CATALOG OF FEDERAL DOMESTIC ASSISTANCE NUMBER

TITLE:

11. DESCRIPTIVE TITLE OF APPLICANT'S PROJECT*

Development of a Novel Inhibitor of Ricin: A Potential Therapeutic Lead against Deadly Shiga and Related Toxins

12. PROPOSED PROJECT

Start Date* Ending Date*

04/01/2017 03/31/2020

13. CONGRESSIONAL DISTRICTS OF APPLICANT

NY-010
Contact PD/PI: Domashevskiy, Artem

Tracking Number: GRANT12199590 Funding Opportunity Number: PA-16-200 . Received Date:

2016-06-22T16:33:35.000-04:00

SF 424 (R&R) APPLICATION FOR FEDERAL ASSISTANCE Page 2

14. PROJECT DIRECTOR/PRINCIPAL INVESTIGATOR CONTACT INFORMATION

Prefix: First Name*: Artem Middle Name: Last Name*: Domashevskiy Suffix: Ph.D

Position/Title: Assistant Professor

Organization Name*: Research Foundation of CUNY o/b/o John Jay College

Department: Sciences

Division:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

ZIP / Postal Code*:

Phone Number*: Fax Number: Email*: 

15. ESTIMATED PROJECT FUNDING

a. Total Federal Funds Requested* $

b. Total Non-Federal Funds* $0.00

c. Total Federal & Non-Federal Funds* $

d. Estimated Program Income* $0.00

16. IS APPLICATION SUBJECT TO REVIEW BY STATE

EXECUTIVE ORDER 12372 PROCESS?*

a. YES ❍ THIS PREAPPLICATION/APPLICATION WAS MADE

AVAILABLE TO THE STATE EXECUTIVE ORDER 12372

PROCESS FOR REVIEW ON:

DATE:

b. NO ● PROGRAM IS NOT COVERED BY E.O. 12372; OR

❍ PROGRAM HAS NOT BEEN SELECTED BY STATE FOR

REVIEW

17. By signing this application, I certify (1) to the statements contained in the list of certifications* and (2) that the statements herein

are true, complete and accurate to the best of my knowledge. I also provide the required assurances * and agree to comply with

any resulting terms if I accept an award. I am aware that any false, fictitious, or fraudulent statements or claims may subject me to

criminal, civil, or administrative penalties. (U.S. Code, Title 18, Section 1001)

● I agree*

* The list of certifications and assurances, or an Internet site where you may obtain this list, is contained in the announcement or agency specific instructions.

18. SFLLL or OTHER EXPLANATORY DOCUMENTATION File Name: 

19. AUTHORIZED REPRESENTATIVE

Prefix: Ms. First Name*: Susy Middle Name: G. Last Name*: Mendes Suffix: 

Position/Title*: Director

Organization Name*: Research Foundation of CUNY o/b/o John Jay College

Department: Sponsored Programs

Division:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

ZIP / Postal Code*:

Phone Number*: Fax Number: Email*: 

Signature of Authorized Representative*

Susy Mendes

Date Signed*

06/22/2016

20. PRE-APPLICATION File Name: Mime Type:

21. COVER LETTER ATTACHMENT File Name:1241-Cover_Letter_ADomashevskiy.pdf Mime Type: application/pdf
424 R&R and PHS-398 Specific

Table Of Contents Page Numbers

SF 424 R&R Cover Page----------------------------------------------------------------------------------------- 1

 Table of Contents------------------------------------------------------------------------- 3

Performance Sites--------------------------------------------------------------------------------------------- 4

Research & Related Other Project Information------------------------------------------------------------------ 5

 Project Summary/Abstract(Description)----------------------------------------------------- 6

 Project Narrative------------------------------------------------------------------------- 7

 Facilities & Other Resources-------------------------------------------------------------- 8

 Equipment--------------------------------------------------------------------------------- 12

Research & Related Senior/Key Person-------------------------------------------------------------------------- 14

Research & Related Budget Year - 1---------------------------------------------------------------------------- 20

Budget Justification------------------------------------------------------------------------------------------ 23

Research & Related Cumulative Budget-------------------------------------------------------------------------- 26

PHS398 Cover Page Supplement---------------------------------------------------------------------------------- 27

PHS 398 Research Plan----------------------------------------------------------------------------------------- 29

 Introduction to Application--------------------------------------------------------------- 30

 Specific Aims----------------------------------------------------------------------------- 31

 Research Strategy------------------------------------------------------------------------- 32

 Bibliography & References Cited----------------------------------------------------------- 44

 Letters of Support------------------------------------------------------------------------ 50

 Authentication of Key Biological and/or Chemical Resources-------------------------------- 56

Contact PD/PI: Domashevskiy, Artem

Table of Contents Page 3
Contact PD/PI: Domashevskiy, Artem

OMB Number: 4040-0010

Expiration Date: 06/30/2016

Tracking Number: GRANT12199590 Funding Opportunity Number: PA-16-200. Received Date:

2016-06-22T16:33:35.000-04:00

Project/Performance Site Location(s)

Project/Performance Site Primary Location ❍ I am submitting an application as an individual, and not on behalf of

a company, state, local or tribal government, academia, or other type of

organization.

Organization Name: John Jay College of Criminal Justice

Duns Number:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

Zip / Postal Code*:

Project/Performance Site Congressional District*:

File Name Mime Type

Additional Location(s)

 Page 4
Tracking Number: GRANT12199590 Funding Opportunity Number: PA-16-200. Received Date:

2016-06-22T16:33:35.000-04:00

OMB Number: 4040-0001

Expiration Date: 06/30/2016

RESEARCH & RELATED Other Project Information

1. Are Human Subjects Involved?* ❍ Yes ● No

1.a. If YES to Human Subjects

 Is the Project Exempt from Federal regulations? ❍ Yes ❍ No

If YES, check appropriate exemption number: 1 2 3 4 5 6

If NO, is the IRB review Pending? ❍ Yes ❍ No

IRB Approval Date:

Human Subject Assurance Number

2. Are Vertebrate Animals Used?* ❍ Yes ● No

2.a. If YES to Vertebrate Animals

Is the IACUC review Pending? ❍ Yes ❍ No

IACUC Approval Date:

Animal Welfare Assurance Number

3. Is proprietary/privileged information included in the application?* ❍ Yes ● No

4.a. Does this project have an actual or potential impact - positive or negative - on the environment?* ❍ Yes ● No

4.b. If yes, please explain:

4.c. If this project has an actual or potential impact on the environment, has an exemption been authorized or an

environmental assessment (EA) or environmental impact statement (EIS) been performed?

❍ Yes ❍ No

4.d. If yes, please explain:

5. Is the research performance site designated, or eligible to be designated, as a historic place?* ❍ Yes ● No

5.a. If yes, please explain:

6. Does this project involve activities outside the United States or partnership with international

collaborators?*

❍ Yes ● No

6.a. If yes, identify countries:

6.b. Optional Explanation:

Filename

7. Project Summary/Abstract* 1236-Abstract_ADomashevskiy.pdf Mime Type: application/pdf

8. Project Narrative* 1237-

Public_Health_Relevance_Statement.pdf

Mime Type: application/pdf

9. Bibliography & References Cited 1238-

Bibliography_ADomashevskiy.pdf

Mime Type: application/pdf

10.Facilities & Other Resources 1239-

Facilities_and_Other_Resources.pdf

Mime Type: application/pdf

11.Equipment 1240-

Equipment_ADomashevskiy.pdf

Mime Type: application/pdf

Contact PD/PI: Domashevskiy, Artem

 Page 5
Abstract

The PI proposes a high-impact collaborative research project to develop new inhibitors for ribosome 

inactivating proteins (RIPs), such as ricin and Shiga toxin. Ricin toxin, produced by the castor bean plant, has a 

nefarious past. Ricin is a well-known homicidal poison and has been used in several bioterrorist attacks. Shiga 

toxin is a deadly product of enterobacterial Escherichia coli. RIPs are RNA N-glycosidases that enzymatically 

remove specific purine residues from the universally conserved sarcin/ricin loop (S/R loop) of large ribosomal 

RNA, causing cellular death. There are no effective pharmaceuticals for either ricin or Shiga toxin poisoning. 

The PI proposes using a viral protein (VPg) from turnip mosaic virus that we have shown to inhibit ricin

activity in vitro. The goals of this proposal are: 1) Establish conditions for VPg-ricin complex formation and the 

rates of VPg-ricin interactions; 2) Determine minimum VPg peptide that effectively inhibits ricin and Shiga 

toxins; and 3) Optimize conditions to enhance inhibition of these toxic proteins. This is an innovative activity. 

Based on the structural and mechanistic similarities of these toxins and previously acquired data showing that

VPg peptides inhibit ricin activity in vitro, we believe the outcomes of this activity will provide leads for the 

synthesis of therapeutic peptides. In turn, this will serve as a catalyst for the development of constructively

applied solutions for the inhibition of these deadly toxins. Furthermore, this proposal will enhance the 

infrastructure of research and education at John Jay College, introducing biochemical and biomedical research

experiences to underrepresented minority and female students, who would otherwise lack such opportunities. 

This would allow them to experience a broad spectrum of techniques, and acquire skills such as data analysis 

used in modern scientific investigations, while developing a vast network of partnerships among scientists from 

national and international institutions.

Contact PD/PI: Domashevskiy, Artem

Project Summary/Abstract Page 6
Public Health Relevance Statement 

Ricin and related Shiga toxins are potentially lethal poisons, which as of yet, have no effective pharmaceutical 

treatments or preventions. The global problem of these poisons is an increasing threat to public health, directly 

relating to the mission of the National Institutes of Allergy and Infectious Diseases. Ricin, astoundingly easily 

acquired, is an ideal toxin for bioterrorism, while the widespread transmission of E. coli poisoning is responsible 

for many severe human illnesses and several fatalities. In the future, it may be possible to utilize these toxins for 

their public health benefit, by repurposing them to attack specific human and animal diseases. 

Contact PD/PI: Domashevskiy, Artem

Project Narrative Page 7
Facilities and Other Resources

Student Profile

In the last year, John Jay had a total enrollment of 12,969 undergraduates and 1,763 graduate students. Of the 

14,732 enrolled students, 20% identify as African American/Black and nearly 41% identify as Hispanic. Nearly 

57% of the student body is female. (John Jay College, Office of Institutional Research, Fall 2015).

Institutional Characteristics

John Jay College is designated as both a Minority and Hispanic-Serving Institution by the U.S. Department of 

Education and as a Primarily Undergraduate Institution by the National Science Foundation. Many of our 

students are eager to take advantage of opportunities in research and academia. The College offers 

baccalaureate and advanced degrees in the biomedical and behavioral sciences, e.g. forensic psychology, 

forensic science, cell and molecular biology, toxicology, etc.

Laboratory

Dr. Domashevskiy (the PI) directs a research laboratory (05.65 & 05.69 New Building) specifically tailored to

his research interests. In addition to Dr. Domashevskiy access to a personal computer and printer, John Jay 

College is equipped with printers for producing large size posters for conference presentations. The College 

provides centralized computing services through the offices of the Department of Information Technology 

(DoIT). This Department supports a comprehensive range of computing, networking, file transfer and data 

communication services, as well as access to computer-related equipment, such as optical scanners, laser 

printers, etc. DoIT staff provides sophisticated programming and consultation. John Jay currently supports 

wireless access at several locations throughout its campus.

Office

The PI also has an office, located at 05.66.25 New Building of John Jay College, 524 West 59th St., New York, 

NY 10019. In 2012, the College completed construction on this new 620,000-square-foot building, 13-story 

vertical campus, that houses 56 state-of-the-art classrooms, new cyber lounges, computer labs, cutting edge 

science facilities, Emergency Management and High Rise Simulator labs, conference rooms, an exhibit gallery, a 

black box theater, great dining facilities and many other educational and administrative features in a 100 percent 

wireless environment. This office is hardwired to the CUNY system, including the library. 

Other Assets 

Resources typically found as part of a major liberal arts college are available at John Jay College. The College 

and CUNY provide state-of-the-art library resources. All faculties have onsite and remote online access to the 

CUNY+ library system which provides access to the millions of books and thousands of periodicals held by 

the 18 libraries of CUNY as well as New York City’s public libraries. The Lloyd Sealy Library on the campus at 

John Jay itself holds more than 300,000 books, periodicals, and microforms. The CUNY+ library system 

provides online access to over 150 online databases including Medline, PsychInfo, Sociological Abstracts, and 

Anthropological Abstracts, and to thousands of online periodicals. CUNY+ maintains a membership in the 

Inter-University Consortium for Political and Social Research (ICPSR) and provides access to ICPSR data files 

without charge to CUNY faculty and advanced students engaged in social science research. CUNY+ maintains 

affiliations with many New York area libraries and provides an Inter-Library Loan service for books and 

periodicals that are not available within the CUNY system. In addition to library services, John Jay College also 

maintains excellent audiovisual facilities, a duplicating and print shop, and a telecommunications system that 

enables faculty and collaborators to communicate electronically and to retrieve bibliographic sources online. 

Student Research Programs

The most developed student research program at John Jay College is the Program for Research Initiatives in 

Science and Math (PRISM). PRISM engages several hundred students annually in lectures, workshops, and 

outings centered on fostering interest in professional careers in science. PRISM also organizes a research 

Contact PD/PI: Domashevskiy, Artem

Facilities & Other Resources Page 8
training course twice per year and funds more than 40 students per term (fall, spring, and summer) in mentored 

research relationships with faculty. The program provides travel funding for students and mentors to academic 

conferences, and supports the acquisition of equipment and supplies related to undergraduate research by 

PRISM faculty. The program organizes an annual research symposium at which some 40 students give oral or 

poster presentations to a college-wide audience. With almost two dozen faculty members participating, the 

program prides itself on deep mentoring, fostering multi-year relationships between students and faculty that not 

only improve retention and graduation, but result in significant scientific achievements for our students. The 

rate of presentation at national research conferences or co-authorship on peer-reviewed publications in our 

program far exceeds rates identified in the literature for other research-training programs (Table 1).

Table 1. Statistics for student success at John Jay College.

Thiry at al. (2012)

n = 73

Hunter et al. (2006)

n = 76

Chaplin et al. (1998)

n = 47

PRISM

n = 101

% Students w/ Academic 

Presentations

10% 9.2% 17.2% 38.2%

% Students with 

Publication Authorship

10% 6.6% NR 28.2%

Founded in the early 2000s, PRISM has been highly effective at improving the success and post-graduate 

careers of students in sciences. The program has resulted in a 5-fold increase in the number of students moving 

on to Ph.D./M.D. degrees in math and science fields. This led to recognition in 2011 by the National Science 

Foundation with a Presidential Award for Excellence in Science, Math, and Engineering Mentoring given by 

President Barack Obama.

The College also maintains other mentoring programs. The College participates in McNair and NSFSTEP programs for supporting research among STEM students, and a Vera Foundation funded program 

supports student scholars focused broadly on issues of justice or disparity. In addition, the College operates a 

central Office of Undergraduate Research to coordinate, disseminate, and strengthen efforts aimed at 

undergraduate research mentoring, which was also launched with grant funding from the U.S. Department of 

Education. The Office organizes a week-long Celebration of Student Research and Creativity Event which 

brings together all students at the College in a series of lectures, poster presentations, performances, and other 

events that further promote and showcase student research activities at the College. 

Speaker Series/Seminars

The College maintains numerous seminar series for the professional development of our students. STEM 

lecture series are organized at the College both by our science masters programs and by a Ph.D. program 

housed at the Graduate Center of the City University of New York. Psychology maintains a separate research 

seminar series specifically for topics in that field. PRISM organizers monthly meetings with professionals who 

come speak to our students about entry into professional STEM graduate programs. And finally, multi Collegewide seminars exist that include biomedical and behavioral health topics such as the changing landscape of 

drug abuse in the 21st century, the biological effects of lead exposure on transport system in the brain, and the 

effect of incarceration on major issues of public health to name but a few. 

Institutional Scientific Collaborations and Partnerships

John Jay College actively fosters and promotes relationships with Community Colleges in the sciences through 

the CUNY Justice Academy. These unique partnerships between John Jay and York, Queensborough, Bronx, 

Borough of Manhattan, and Hostos Community Colleges go beyond traditional articulation programs, in that 

admitted students are considered to be enrolled both at John Jay and the two-year partner from the point of 

admission. The program thus engages students in the senior college community at the point of admission, and 

the College invests significant resources in fostering the growth and successful completion of these students 

from their first year.

Contact PD/PI: Domashevskiy, Artem

Facilities & Other Resources Page 9
Currently, some 600+ students are in the community college partnership program with John Jay in 

STEM fields. Given that all of our community college partners are MSIs, and both Hostos and Bronx 

Community are leading 2-year HSIs, a large portion of these students hail from traditionally underrepresented 

groups in the STEM fields. 

John Jay faculty members also maintain extensive relationships with post-baccalaureate training 

institutions. Over half of our science faculty and more than two-thirds of our psychology faculty serve as active 

members of doctoral programs and doctoral dissertation committees at the CUNY Graduate Center. Many of 

our faculty members maintain active collaborations at doctoral institutions in the metropolitan area including 

Columbia University, Mount Sinai Medical School, Albert Einstein College of Medicine, New York University, 

and others. The STEM programs at John Jay have also negotiated formal relationships with the University of 

Lincoln (U.K.) and a burgeoning EU-funded Erasmus Mundus consortium in which students from these 

campuses come to our campus to complete their thesis projects. 

Early State Investigator Support

John Jay College provides myriad support opportunities for the success of its faculty members. Early 

stage investigators are of particular interest. The University Chancellor, the College President and the Provost 

have pledged to fund “The Decade of Science” at CUNY, and have supported the expansion of research 

programs by offering competitive lab start up packages and increasing the allocation of space to construct new 

labs. Also, grant holders are able to purchase release time at the less expensive replacement time rate, thus 

resulting in savings to the grant agencies. The College has provided an additional incentive to match grant 

purchased course release time. Official start-up packages were first presented in 2006, and offers have increased 

since then. The College also provides all the necessary support and resources for classes, and holds frequent 

teaching and training programs to enrich faculty pedagogical approaches, advancement in career, assistance and 

guidance in the mentorship and supervision of the personnel involved in the proposed activity. 

The Office for Advancement of Research (OAR) at John Jay College is dedicated to working with 

faculty in the pursuit of their research and scholarly goals, and is available to assist faculty in grant-seeking and 

publication activities. Faculty members are encouraged to consult with OAR on any aspect of these processes. 

OAR promotes faculty success through several funding programs, including: The Emergency Fund program 

(support is available for research or other scholarly activities that specifically address a need that is urgent 

and/or time sensitive in nature), See Money Requests (support pilot or preliminary work necessary to pursue a 

major study to gather background data toward supporting a major proposal), Proposal Peer Review (funds are 

available for faculty to obtain external reviews of grant proposals prior to their submission to an agency or 

funding organization), Open-Access Publication Funding (in an effort to broaden the dissemination of research 

of John Jay scholars, funds are available to faculty who wish to publish articles in open-access format in major, 

peer-reviewed, scholarly journals), Book Publication Funding (support faculty authors who are in the process 

of publishing books), and Enhanced Travel Funding (provides additional travel opportunities to the 

Department’s allocations for travel for faculty to present their research at scientific conferences.

The Program for Research Initiatives for Science and Math (PRISM) at the Department is committed 

to providing its research faculty, mentoring PRISM students, with annual supplies and equipment funds.

Students participating in the PRISM program engage in mentored-supervised research, receive stipend support, 

and present their work at college, state and countrywide symposiums. PRISM increased student participation 

over 60%. Thus, John Jay has a firm commitment to increase research productivity.

The PI’s collaborators have offered their full support and experience in this activity. Dr. Dixie Goss 

(Hunter College, CUNY) has offered her laboratory space, her time, and instrumentation to perform 

fluorescence titration experiments requiring high lamp intensity. Her lab will assist in recombinant protein 

purifications, as well as kinetic studies. Dr. Vern Schramm (Albert Einstein College of Medicine) has offered 

his full expertise in the proposed activity, and has supplied our laboratory with recombinant ricin and other 

proteins used in catalytic assays. Dr. John Latham (Denver University) will devote his time to conduct 

isothermal calorimetry experiments that will identify important thermodynamic parameters of ricin-inhibitor 

interactions. Dr. David Jeruzalmi (City College, CUNY) is an expert in X-ray crystallography, and will aid in 

protein crystallization, diffraction, and data analysis. Dr. Alison O’Brien (Uniformed Services University of 

Contact PD/PI: Domashevskiy, Artem

Facilities & Other Resources Page 10
Health Sciences) has gladly agreed to perform in vivo experiments that will test our central hypothesis that VPg 

will inhibit RIP activity of RTA and Shiga toxin in Vero cells. Dr. Nancy Greenbaum (Hunter College, CUNY) 

has offered her aid in the development of experiments that involve NMR spectroscopy and data analysis. Dr. 

Kathi Hudak (York University, Ontario, Canada) will provide her expert advice and protocols for primer 

extension and determination of depurination sites. Drs. Shaneen Singh and Emilio Gallicchio (Brooklyn 

College) will provide their expertise and knowledge regarding molecular modeling, docking and scoring, and 

will aid in mapping the binding interactions between RTA and VPg. 

The Department of Sciences at John Jay College provides its research faculty with safe facilities to 

conduct their research that may involve potentially toxic and biohazardous substances. Biosafety cabinets and 

fume hoods are available in the PI’s laboratory. All faculty and students involved in experimental research 

participate in mandatory annual seminar/training on Hazardous Waste Disposal. Additional seminars on 

Biohazardous Substances, Radioactivity, etc. are also available to those researchers involved with them. Finally, 

all key personnel have satisfied RCR and COI training. Experiments involving radioactivity (primer extension 

assay) will be conducted at Dr. Goss laboratory at Hunter College. The Department of Sciences at John Jay is 

in the process of acquiring their radioactivity license, expected by the end of 2016. Dr. O’Brien’s laboratory at 

the Uniformed Services University of Health Sciences is fully equipped to handle Shiga toxin and cytotoxic 

ricin, and will perform all the experiments involving these toxins in live cells.

Contact PD/PI: Domashevskiy, Artem

Facilities & Other Resources Page 11
Equipment at John Jay College

The Department of Sciences at John Jay College comprises three floors in a three-year old state of the art 

building on 59th Street. Spread across these three floors are 15,405 feet2 of space dedicated to research 

laboratories and 29,501 feet2 dedicated to teaching laboratories. There are 27 biosafety cabinets in the 

department, 77 fume hoods, three full-sized and two table-top autoclaves, three water purification systems, two 

glassware facilities, three microscopy suite, each for different types of microscopy (fluorescent, FT-IR, and a 

scanning electron microscopy facility), and a nuclear magnetic resonance (NMR) facility with a 300MHz 

magnet. There are three cell culture facilities dedicated as such with additional cell culture work taking place as 

well.

Major shared instrumentation existing in core shared research space (located 30 feet away PI’s laboratory)

include five -80oC freezers, one Sorvall floor centrifuge, one Beckman ultracentrifuge, and one X-series ICPMS. The following equipment exists across three dual-use teaching/research facilities and are available for 

faculty to use for research: Three GC/FID instruments, one fluorescence spectrometer, one X-ray 

diffractometer, three HPLC units, four GC-MS units (including one triple quadripole unit), two atomic 

absorption spectrometers, five GC units, three microplate readers, one LC-MS instrument, three FT-IR 

spectrometers, one Luminex autoplex analyzer, three Agilent Bioanalyzers, two ABI 7500 real-time PCR units, 

one Life Technologies 3500 Genetic Analyzer, and six nano-drop 2000C spectrophotometers. 

We include here the major equipment inventories of several teaching and research laboratories of 

individual faculty members: 

Dept. of Sciences – Teaching Laboratory Equipment and Instrumentation

Course Description Quantity

Chemistry THERMO SCIENTIFIC GENESYS 20 UV SPECTROPHOTOMETER 18

Biology MICROSCOPE OLYMPUS CHT 24

MICROSCOPE LEICA STRATA LAB 10

WALTER STEREO MICROSCOPE 16

E-GEL IBASE 10

INVITROGEN E-GEL SAFE IMAGER 4

MONOLUXHD WIFI MICROSCOPE IPAD 16

Toxicology TECAN MICROPLATE READER 2

THERMO TRACE DSQ - MS 1

THERMO TRACE GC ULTRA 1

AGILENT HPLC 1100 2

AGILENT 6890N GC SYSTEM 1

SHIMADZU UV-2600 UV/VIS SPECTROPHOTOMETER 3

BIOTAGE TURBOVAP LV EVAPORATION SYSTEM 1

TRIPLE QUADRIPOLE LCMS SYSTEM - SHIMADZU LCMS-8030 

SYSTEM

1

Molecular HYBRIDIZATION INCUBATOR 1

Biology REVCO ULTRA LOW FREEZER 1

PERKIN ELMER/ LUMINEX CS 1000 AUTOPLEX ANALYZER + 

STAND

1

AGILENT 2100 BIOANALYZER 2

ABI GENE AMP PCR 9700 2

ABI 7500 REAL TIME PCR SYSTEM 2

LIFE TECHNOLOGIES 3500 GENETIC ANALYZER 1

AMERSHAM ULTROSPEC 1100 PRO UV SPECTROPHOTOMETER 13

 

Contact PD/PI: Domashevskiy, Artem

Equipment Page 12
Faculty Mentor Description of Equipment

Dr. 

Domashevskiy AKTA PURE PROTEIN PURIFIER SYSTEM

HORIBA JOBIN YVON FLUOROMAX-3 

BIORAD HELIOS GENE GUN & PREP STATION

SHIMADZU UV-VIS SPECTROPHOTOMETER

GENO GRINDER, TISSUE HOMOGENIZER

PHAST SYSTEM ELECTROPHORESIS

EPPENDORF REFRIGERATED MICROFUGE

WATERS HPLC WITH ABSORBANCE DETECTOR, FLUORESCENCE 

DETECTOR, CONTROLLER AND AUTOSAMPLER

Dr. Rauceo REAL TIME PCR DETECTION SYSTEM

CARL ZEISS DISSECTING MICROSCOPE OR STEREO MICROSCOPE

EPPENDORF MASTERCYCLER EPGRADIENT S

EPPENDORF EPGRADIENT S MASTERCYCLER

QIAGEN TISSUELYSER II

NANODROP SPECTROPHOTOMETER

Dr. Carpi TEKRAN 2537B MERCURY VAPOUR ANALYZER

OCEANAIRE AIR BOSS AC

MICROSCOPE NIKON ECLIPSE E600

PERKIN ELMER THERMAL CYCLER 480

MERCURY TEKRAN VAPUOR ANALYZER 2537A

Dr. Lents PERKIN ELMER GELIANCE 200

PERKIN ELMER GELIANCE 600

EPPENDORF MASTERCYCLER EPGRADIENT 

AB APPLIED BIOSYSTEM 2720 THERMALCYCLER

TURNER BIOSYSTEMS LUMINOMETER

AGILENT 2100 BIOANALYZER

APPLIED BIOSYSTEM 7500 REAL TIME PCR SYSTEM

AIRCLEAN PCR WORKSTATION 600

BIORAD CHEMIDOC MP IMAGING SYSTEM

BIORAD TC20 AUTOMATED CELL COUNTER

THERMOFISHER NANODROP SPECTROMETER

Dr. Kobilinsky 0700 XES CONTROL X-Ray Unit

PANASONIC WV-BL200

DELTASPHERE IN IM2720 CASE

NIKON MICROSCOPE WITH CAMERA

NIKON FX-35

BIORAD BENCHMARK PLUS MICROPLATE SPECTROPHOTOMETER

Dr. Yaverbaum TOBII TX300 EYETRACKER

Dr. Wallace AB APPLIED BIOSYSTEM GENEAMP PCR SYSTEM 2400

310 GENETIC ANALYZER

Dr. Cheng APPLIED BIOSYSTEMS CYTOMETER - ATUNE

INVERTED TISSUE CULTURE MICROSCOPE

EPPENDORF REFRIGERATED MICROFUGE

SANYO CO2 INCUBATOR

Contact PD/PI: Domashevskiy, Artem

Equipment Page 13
OMB Number: 4040-0001

Expiration Date: 06/30/2016

Tracking Number: GRANT12199590 Funding Opportunity Number: PA-16-200 . Received Date:

2016-06-22T16:33:35.000-04:00

RESEARCH & RELATED Senior/Key Person Profile (Expanded)

PROFILE - Project Director/Principal Investigator

Prefix:  First Name*: Artem Middle Name  Last Name*: Domashevskiy Suffix: Ph.D

Position/Title*: Assistant Professor

Organization Name*: Research Foundation of CUNY o/b/o John Jay College

Department: Sciences

Division:

Street1*:

Street2:

City*:

County:

State*:

Province:

Country*:

Zip / Postal Code*:

Phone

Number*: 

Fax Number:  E-Mail*: 

Credential, e.g., agency login: 

Project Role*:  PD/PI Other Project Role Category: 

Degree Type:  PhD Degree Year:  2011

File Name Mime Type

Attach Biographical Sketch*: 1235-

Biosketch_ADomashevskiy.pdf

application/pdf

Attach Current & Pending Support:

Contact PD/PI: Domashevskiy, Artem

 Page 14
Contact PD/PI: Domashevskiy, Artem

OMB No. 0925-0001 and 0925-0002 (Rev. 10/15 Approved Through 10/31/2018) 

BIOGRAPHICAL SKETCH

Provide the following information for the Senior/key personnel and other significant contributors. 

Follow this format for each person. DO NOT EXCEED FIVE PAGES.

NAME: Domashevskiy, Artem V.

eRA COMMONS USER NAME (credential, e.g., agency login): 

POSITION TITLE: Assistant Professor of Biochemistry 

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include 

postdoctoral training and residency training if applicable. Add/delete rows as necessary.)

INSTITUTION AND LOCATION

DEGREE

(if applicable)

Completion 

Date

MM/YYYY

FIELD OF STUDY

Hunter College, City University of New York B.A. 01/2006 Chemistry

Graduate Center, City University of New York M.A. 05/2009 Biochemistry

Graduate Center and Hunter College, CUNY Ph.D. 06/2011 Biochemistry

John Jay College of Criminal Justice, CUNY Postdoctoral 08/2012 Biochemistry/Biophysics

A. Personal Statement

I possess the knowledge and expertise required to execute the proposed research project with the motivation, 

devotion and leadership necessary to see it to a successful end. I have a broad background in biochemistry, 

molecular biology and biophysics, with specific training in eukaryotic and viral translation initiation, gene and 

protein expression, kinetic assay analysis and fluorescence. My research involves plant protein toxins known as 

ribosome inactivating proteins (RIPs), specifically pokeweed antiviral protein (PAP) isolated from common 

pokeweed plant (Phytolacca americana), with the goal of exploring and utilizing its antiviral properties. My current 

research entails the detailed study of related RIPs: ricin and Shiga toxin. As a PI, I have developed a mature 

research program involving undergraduate and graduate students. My research is highly collaborative; having 

laid the groundwork for the proposed research by developing vast networks of relationships with local, national 

and international researchers. I mentor and supervise the students, stress awareness of timelines for research, 

foster effective communication, and prepare them to perform research of their own after attaining graduate 

degrees in their chosen fields. The current application builds upon my previous research. 

1. Domashevskiy, A.V., Miyoshi, H. and Goss, D.J. (2012) Inhibition of Pokeweed Antiviral Protein 

(PAP) by Turnip Mosaic Virus Genome-Linked Protein (VPg). J. Biol. Chem. 287, 29729-29738.

2. Domashevskiy, A.V. and Goss, D.J. (2015) Pokeweed Antiviral Protein (PAP), a Ribosome Inactivating 

Protein: Activity, Inhibition and Prospects. Toxins (Basel) 7, 274-298.

3. Domashevskiy, A.V. and Cheng, S.Y. (2015) Thermodynamic Analysis of Binding and Enzymatic 

Properties of Pokeweed Antiviral Protein (PAP) toward Tobacco Etch Virus (TEV) RNA. J. Nat. Sci. 1, 

e82. ISSN: 2377-2700.

4. Aitbakieva, V.R. and Domashevskiy, A.V. (2016) Insights into the Molecular Antiviral Mechanism of 

Pokeweed Protein from Phytolacca americana. Biochem. Pharmacol (Los Angel) 5, 210. 

Biosketches Page 15
B. Positions and Honors

Positions and Employment

2006-2011 Adjunct Lecturer, Department of Chemistry, Hunter College, New York, NY

2008-2012 Lecturer, Department of Sciences, John Jay College, New York, NY

2011-2012 Adjunct Assistant Professor, Department of Chemistry, Hunter College, New York, NY 

2011-2012 Adjunct Assistant Professor, Department of Sciences, John Jay College, New York, NY 

2012 Adjunct Assistant Professor, New York Institute of Technology, New York, NY 

2012-Present Assistant Professor, Department of Sciences, John Jay College, New York, NY

Other Experience and Professional Memberships

2006-Present Member, American Chemical Society (ACS)

2006-Present Member, Undergraduate Poster Judge, American Society of Biochemistry and Molecular 

Biology (ASBMB)

2006-Present Member, New York Academy of Sciences

2012-Present Research Poster Judge, New York City Science and Engineering Fair (NYCSEF)

2012-Present Member, Undergraduate Poster Judge, American Plant Biology Society (ASPB)

2013-Present Reviewer, Division of Biological and Earth Sciences, Professional Staff Congress (PSC), CUNY

2014-Present Member, American Phytopathological Society (APS)

2014-Present Member, Biophysical Society

2014-Present Member, The RNA Society

2014 Program Committee Member, Forensic Science Criminalistics Research (FSCR)

2015-Present Reviewer, Toxins

2015-Present Reviewer, Biophysica et Biochimica Acta

2015-Present Editorial Board, Jacobs Journal of Molecular and Translational Medicine 

2015-Present Editorial Board, Journal of Pharmaceutical Sciences and Emerging Drugs (JPSEM)

2015-Present Editorial Board, Journal of Forensic Science and Legal Medicine 

2015-Present Reviewer, Division of Chemistry and Biochemistry, Professional Staff Congress (PSC), CUNY

 

Honors

2002-2011 Julianne B. Fusco Scholarship, Dynamite Youth Foundation, Inc. Brooklyn, NY 

2006 Helen S. Schectman Award, Hunter College, CUNY, New York, NY

2006-2007 Science Fellowship, Graduate Center, CUNY, New York, NY

2007-2011 CUNY Fellowship, Graduate Center, New York, NY

2015-2016 William Stewart Award, CUNY Academy for the Humanities and Sciences, New York, NY

2016 Henry Wasser Award, CUNY Academy for the Humanities and Sciences, New York, NY

C. Contribution to Science

1. My early research involved investigations into plant viral protein synthesis. Plant viral diseases affect a 

significant number of food crops world-wide, and can have severe impact on economic conditions and crop 

yield. Protein synthesis is a key step in viral infection, yet is not well understood. The research involved 

interactions of translation initiation factors with a set of viral RNAs of varying secondary structures. The 

findings (preference of the tobacco etch virus RNA to bind eIF4G rather than eIFiso4G; viral protein recruits

cellular translational components and increases viral protein translation) increased our understanding of plant 

viral protein synthesis, and how this important biochemical process can be regulated. This information can 

potentially be used to shut off viral protein synthesis either through genetic engineering or biochemical agents. 

Furthermore, viral protein synthesis in general is simpler than host protein synthesis, so there is potential to use 

this research data to develop systems that produce desired proteins which are nutritionally beneficial, 

antibodies, or have other economic uses. I directed equilibrium and kinetic studies of the experiments, engaged 

Contact PD/PI: Domashevskiy, Artem

Biosketches Page 16
in recombinant protein purifications and analysis, designed and performed transcriptional and translational 

assays.

a. Ray, S., Yumak, H., Domashevskiy, A., Gallie, D.R. and Goss, D.J. (2006) Tobacco Etch Virus mRNA 

Preferentially Binds Wheat Germ Eukaryotic Initiation Factor (eIF)4G rather than (eIF)iso4G. J. Biol. 

Chem. 281, 35826-35834.

b. Goss, D.J., Khan, M.A., Domashevskiy, A.V. and Yumak, H. (2008) Biophysical Insights into the 

Mechanism of Viral Protein Synthesis. MARM ACS J. 40, 254.

c. Goss, D.J. and Domashevskiy, A.V. (2015) Messenger RNA (mRNA): The Link between DNA and 

Protein. Encyclopedia of Cell Biology. Vol. 1 pp. 341-345. Academic Press, Eds. Bradshaw, R.A. and Stahl, 

P.D.

d. Domashevskiy, A.V. (2016) Turnip Mosaic Virus Genome-Linked Protein May Confer Resistance to 

Plant Defense Ribosome Inactivating Protein from Phytolacca americana. Virol Mycol. Submitted; under 

reviews.

2. Additionally, I have developed synthesis of a novel fluorescent 5’-cap analog (Anthranioyl-m7GpppG), and 

a novel method for labeling messenger RNA with this fluorescent probe. Nucleic acids have poor intrinsic 

fluorescence, and, other extrinsic fluorophores must be employed to study nucleic acid molecular dynamics and 

structure. The molecule, Ant-m7GTP could be specifically incorporated into the cap site to yield Antm7

GpppG-capped mRNA or oligonucleotide. What makes this extrinsic fluorophore unique and valuable in 

research is a combination of high synthesis yield, efficiency in RNA capping and unique properties of the 

fluorophore itself (absorbance and emission maxima of this probe is far removed from native protein 

fluorescence). This fluorescent probe and the method of capping developed, has a great potential for studies of 

nucleic acid structure and function, their interactions with proteins, and possibly imaging. The Ant-m7GTPcapped RNA provides an important tool for monitoring capping reactions, translation, and biophysical studies. 

a. Gunawardana, D., Domashevskiy, A.V., Gayler, K.R. and Goss, D.J. (2015) Properties of mRNAs 

containing a novel fluorescent analog: Anthranioyl-m7GpppG. Translation 3, e988538.

b. Rodriguez, D. and Domashevskiy, A.V. (2015) Synthesis of Fluorescently-Labeled Tobacco Etch Virus 

(TEV) RNA and Its Interactions with Pokeweed Antiviral Protein. FASEB J. 29:LB222.

c. Rodriguez, D. and Domashevskiy, A.V. (2016) Interactions of a Fluorescently-Labeled Tobacco Mosaic 

Virus (TMV) RNA with Pokeweed Antiviral Protein (PAP). FASEB J. 30:591.1.

d. Domashevskiy, A.V., Rodriguez, D.J., Gunawardana, D., and Goss, D.J. (2016) Preparation of 

functional, fluorescently labeled mRNA capped with Anthraniloyl-m7GpppG. In Synthetic mRNA: 

Production, Introduction into Cells, and Physiological Consequences. Ed. Rhoads, R. Methods Mol. Biol.

1428, 61-75.

3. A unique aspect of my research experiences are plant protein toxins, known as RIPs. Ricin is a lethal poison 

that has no treatment or prevention. Other RIPs have been explored for their antiviral and antifungal 

properties. Pokeweed antiviral protein isolated from pokeweed plant, is a cap-binding protein and an RNA Nglycosidase that depurinates ribosomes. PAP exerts potent antiviral properties. The antiviral activity of PAP 

differs from its ribosome depurinating activity. PAP lowers infectivity of many plant and animal viruses by 

directly depurinating viral RNA. My research involved interactions of PAP with a small viral genome-linked 

peptide VPg from Turnip Mosaic Virus. VPg is important in many different viral functions. My findings 

showed that VPg binds to PAP with high affinity, and it inhibits PAP enzymatic activity. I believe that VPg 

may be an evolutionary adaptation of the virus against plant defense mechanism – PAP. An evolutionary arms 

race between plants, synthesizing RIPs, and their pathogens that have evolved to produce a small peptide that 

inhibits RIP activity, was characterized in these studies. Antiviral properties of PAP are investigated in my 

laboratory. I am the Principal Investigator on these projects, and have conducted fluorescence studies, 

produced depurination and catalytic activity assays, HPLC serration and quantification. 

a. Aitbakieva, V. and Domashevskiy, A.V. (2015) Characterization of Pokeweed Antiviral Protein (PAP) 

Isoforms and Comparison of Their Enzymatic Activities toward Tobacco Etch Virus RNA. FASEB J.

29, LB130.

Contact PD/PI: Domashevskiy, Artem

Biosketches Page 17
b. Williams, S. and Domashevskiy, A.V. (2014) Pokeweed Antiviral Protein Binds to Structures Present in 

the 3’ Untranslated Regions of Viral mRNA. Annual Biomedical Research Conference for Minority Students. San 

Antonio, TX. 

c. Cheng, S.Y., Domashevskiy, A.V. and Kobilinsky, L. (2014) The Effect of Eukaryotic Initiation Factors 

on the Activity of Pokeweed Antiviral Protein. Society of Toxicology Annual Conference. Phoenix, AZ. 

d. Domashevskiy, A.V. and Goss, D.J. (2013) Overcoming Plant Defense Mechanisms. American Society of 

Plant Biologists Annual Conference. Providence, RI.

4. A novel avenue of my research involves investigation into how male routine infant circumcision (RIC) 

affects the length and degradation of chromosomal telomeres and development of autism as a direct 

consequence of RIC, as well as advocacy for rights of children. This is a highly contagious subject in the 

United States, with much convoluted information. While female circumcision is explicitly forbidden and 

punishable under the current laws of the United States, male circumcision is not. It is considered a nontherapeutic elective surgery legally performed by surrogate consent, violating the basic human rights of 

male children and in direct violation of the US Constitution granting equal protection under the law. 

a. Domashevskiy, A.V. and Domashevskiy, J.A. (2016) Ethics Pertaining to the Legalities of Male Routine 

Infant Circumcision and Surrogate Consent to Non-Therapeutic Surgery. J. Clin. Res. Bioeth. Submitted; 

under reviews.

Complete List of Published Work in My Bibliography:

http://www.ncbi.nlm.nih.gov/sites/myncbi/1p3WpcuErld5c/bibliography/40599058/public/?sort=

date&direction=ascending

D. Research Support

Ongoing Research Support

Professional Staff Congress (PSC), Cycle 47 Role: PI 2016-2017

City University of New York internal grant

Investigating effects of plant antiviral protein on HIV RNA. 

Completed Research Support

Program for Research Initiatives for Science and Math (PRISM) Role: PI May 2016

Equipment Grant to purchase BioRad Helios gene Gun. 

Seed Grant, Office of Advancement of Research (OAR) Role: PI 08/2015-06/2016

John Jay College of Criminal Justice 

Synthesis of a functional, fluorescently-labeled RNA. 

Seed Grant, Office of Advancement of Research (OAR) Role: PI 07/2015-05/2016

John Jay College of Criminal Justice 

Development of a novel inhibitor of ricin. 

Program for Research Initiatives for Science and Math (PRISM) Role: PI June 2015

Equipment Grant to purchase AKTA Pure protein purifier system. 

Program for Research Initiatives for Science and Math (PRISM) Role: co-PI June 2015

Equipment Grant to purchase electroanalytic station (potentiostat).

Contact PD/PI: Domashevskiy, Artem

Biosketches Page 18
Program for Research Initiatives for Science and Math (PRISM) Role: co-PI June 2014

Equipment Grant to purchase Geno/Grinder, a high-throughput put tissue homogenizer. 

Seed Grant, Office of Advancement of Research (OAR) Role: PI 02/2014-02/2015

John Jay College of Criminal Justice 

Targeting HIV with Liposomal Pokeweed Antiviral Protein. 

Professional Staff Congress (PSC), Cycle 44 Role: PI 03/2013 – 06/2014

City University of New York internal grant

Characterization of Enzymatic Mechanism of Pokeweed Antiviral Protein. 

Program for Research Initiatives for Science and Math (PRISM) Role: co-PI June 2014

Equipment Grant to purchase a Circular Dichroism Spectrophotometer. 

Startup Fund Role: PI 08/2012 – 07/2015

John Jay College, CUNY 

Contact PD/PI: Domashevskiy, Artem

Biosketches Page 19
OMB Number: 4040-0001

Expiration Date: 06/30/2016

Tracking Number: GRANT12199590 Funding Opportunity Number: PA-16-200 . Received Date: 2016-06-22T16:33:35.000-04:00

RESEARCH & RELATED BUDGET - SECTION A & B, Budget Period  1

ORGANIZATIONAL DUNS*:   

Budget Type*: ● Project ❍ Subaward/Consortium

Enter name of Organization: Research Foundation of CUNY o/b/o John Jay College

Start Date*: 04-01-2017 End Date*: 03-31-2020 Budget Period: 1

A. Senior/Key Person

# Prefix First Name* Middle

Name

Last Name* Suffix Project Role* Base

Salary ($)

Calendar

Months

Academic

Months

Summer

Months

Requested

Salary ($)*

Fringe

Benefits ($)*

Funds Requested ($)*

1 . Artem Domashevskiy Ph.D PD/PI

Total Funds Requested for all Senior Key Persons in the attached file

Additional Senior Key Persons: File Name: Total Senior/Key Person

Mime Type:

B. Other Personnel

Number of

Personnel*

Project Role* Calendar Months Academic Months Summer Months Requested Salary ($)* Fringe Benefits* Funds Requested ($)*

Post Doctoral Associates

1 Graduate Students 4.50

3 Undergraduate Students 13.50

Secretarial/Clerical

1 Technician 11.82

5 Total Number Other Personnel Total Other Personnel

Total Salary, Wages and Fringe Benefits (A+B)

RESEARCH & RELATED Budget {A-B} (Funds Requested)

Contact PD/PI: Domashevskiy, Artem

 Page 20
Contact PD/PI: Domashevskiy, Artem

Tracking Number: GRANT12199590 Funding Opportunity Number: PA-16-200 . Received Date:

2016-06-22T16:33:35.000-04:00

RESEARCH & RELATED BUDGET - SECTION C, D, & E, Budget Period 1

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium

Organization: Research Foundation of CUNY o/b/o John Jay College

Start Date*: 04-01-2017 End Date*: 03-31-2020 Budget Period: 1

C. Equipment Description

List items and dollar amount for each item exceeding $5,000

Equipment Item Funds Requested ($)*

Total funds requested for all equipment listed in the attached file

Total Equipment

Additional Equipment: File Name:

Mime Type:

D. Travel Funds Requested ($)*

1. Domestic Travel Costs ( Incl. Canada, Mexico, and U.S. Possessions)

2. Foreign Travel Costs

Total Travel Cost

E. Participant/Trainee Support Costs Funds Requested ($)*

1. Tuition/Fees/Health Insurance

2. Stipends

3. Travel

4. Subsistence

5. Other: 

 Number of Participants/Trainees Total Participant Trainee Support Costs

RESEARCH & RELATED Budget {C-E} (Funds Requested)

 Page 21
Contact PD/PI: Domashevskiy, Artem

Tracking Number: GRANT12199590 Funding Opportunity Number: PA-16-200 . Received Date:

2016-06-22T16:33:35.000-04:00

RESEARCH & RELATED BUDGET - SECTIONS F-K, Budget Period 1

ORGANIZATIONAL DUNS*: 

Budget Type*: ● Project ❍ Subaward/Consortium

Organization: Research Foundation of CUNY o/b/o John Jay College

Start Date*: 04-01-2017 End Date*: 03-31-2020 Budget Period: 1

F. Other Direct Costs Funds Requested ($)*

1. Materials and Supplies

2. Publication Costs

3. Consultant Services

4. ADP/Computer Services

5. Subawards/Consortium/Contractual Costs

6. Equipment or Facility Rental/User Fees

7. Alterations and Renovations

Total Other Direct Costs

G. Direct Costs Funds Requested ($)*

Total Direct Costs (A thru F)

H. Indirect Costs

Indirect Cost Type Indirect Cost Rate (%) Indirect Cost Base ($) Funds Requested ($)*

1 . Salaries, wages and fringe benefits

Total Indirect Costs

Cognizant Federal Agency

(Agency Name, POC Name, and POC Phone Number)

DHHS, Ryan Mccarthy, 212-264-2069

I. Total Direct and Indirect Costs Funds Requested ($)*

Total Direct and Indirect Institutional Costs (G + H)

J. Fee Funds Requested ($)*

K. Budget Justification* File Name: 1234-

Budget_Justification_ADomashevskiy rev.pdf

Mime Type: application/pdf

(Only attach one file.)

RESEARCH & RELATED Budget {F-K} (Funds Requested)

 Page 22
Contact PD/PI: Domashevskiy, Artem

Budget Justification

A. Personnel: 

Principal Investigator (1.28 Acad. Months Years 1-3; 2.0 Summer Months Years 1-3) 

The PI will devote himself to the experimental laboratory training of undergraduate and graduate students, 

demonstrate techniques, plan experiments, enforce protocols, and provide assistance with data fitting and 

analysis. He will also be responsible for manuscript preparations and in assisting students with poster 

presentations, by directing group meetings with his research team where practice presentations are 

performed. Additionally, these meetings will present training in basic topics of data search and analysis 

tools, fluorescence, spectroscopy, protein and RNA expression and purification techniques, and poster 

presentations. The group discussions will include current scientific literature and ethics. One course (3 

hours) release and two (2) months of summer salary each year (2/9 of regular academic year salary) are 

requested for Dr. Domashevskiy. The full-time teaching load at John Jay College is 21 hours (7 courses 

teaching/year), thus this request equals 1.28 academic months in each year. He will continue to teach 

biochemistry undergraduate courses and the summer research training programs, but will have gained time 

adequate for the laboratory work, student training, manuscripts preparation, and other responsibilities that 

are directly related to this proposal.

Technician ($ /hr x 15 hrs/wk x 40 weeks) 3.94 Calendar Months each year

Funds are requested for a part-time technician who will be responsible for assisting with undergraduate 

student training in techniques and protocols involving RTA, VPg, and other RIP purification, protein and 

RNA expressions, bacterial growth, and general assistance in overseeing the laboratory, sharing her 

expertise and knowledge, while continuing her own training in the PI’s laboratory. The technician will 

greatly facilitate preparations, assisting in: ordering supplies, maintenance of chemical inventory, and 

compliance with safety regulations. Training of the undergraduate and graduate students is the cornerstone 

of the PI’s research plan. 

Graduate Student ($ /hr x 19 hrs/wk x 12 weeks) 1.5 Calendar Months each year 

Undergraduate Students ($ /hr x 19 hrs/wk x 12 weeks x 3 UG students) 1.5 Calendar Months per UG student 

each year 

Summer support is requested for four undergraduate students and one Masters student, who will learn 

protein and RNA expression and purification, basic biochemistry, perform most of the titration 

experiments, perform depurination and crystallography screening assays and molecular modeling, analyze 

data, and write preliminary drafts of reports, posters, and manuscripts. The MS student will perform 

fluorescence titrations, oversee depurination assay design and methods, and perform extrinsic fluorophore 

protein labeling. All undergraduate students will have a unique part of the proposed Specific Aims assigned 

to them for investigation. 

B. Fringe Benefits: Fringe benefits are calculated by the City University of New York (CUNY) and the 

Research Foundation of CUNY, and are as follows: 

• Release Time: 49%

• Summer Salary: 26%

• Part-time B (working 19 hours/week or less) Project Staff: 9.84%

(A 0.34% MTA tax is included in the calculation for FT/PTA/PTB project staff.)

 

C. Equipment: N/A

D. Travel: The PI requests travel funds of $3,686 for the second and $3,470 for the third year of activity. 

Dr. Domashevskiy and one undergraduate student will attend relevant scientific conferences and will 

present their research as poster/oral presentations. The conferences planned for the team’s attendance will 

include American Society if Plant Biology (ASPB) (July 14-18, 2018; Montreal, Canada), and Biophysical 

Budget Justification Attachment Page 23
Society (February 15-19, 2019; San Diego, CA). Estimated breakdown of the ASPB conference expense 

(US Department of State; Foreign Per Diem Rates): Plane ticket ($275 x 2 = $550), lodging ($179/night x 4 

nights = $716 x 2 = $1,432), meals per Diem ($93 x 4 days = $372 x 2 = $744), local travel ($100 x 2 = 

$200), conference registration fees ($475 + $85 undergrad registration = $560), abstract submission fee 

($60 x 2 = $120), other unexpected expenses ($200) = total $3,806. Estimated breakdown of the 

Biophysical Society conference expense (2016 Domestic Per Diem Rates): Plane ticket ($477 x 2 = $954), 

lodging ($153/night x 4 nights = $612 x 2 = $1,224), meals per Diem ($64 x 4 days = $256 x 2 = $512), 

local travel ($100 x 2 = $200), conference registration fees ($260 + $120 undergrad registration = $380), 

abstract submission fee ($60 x 2 = $120) other unexpected expenses ($200) = total $3,590.

The PI also requests travel funds for the first and second year of activity for him and one 

undergraduate student to visit laboratory of a collaborating researcher. During the first year, the team will 

visit Dr. John Latham (Denver University, CO) to get acquainted with the techniques of isothermal 

titration calorimetry proposed in the activity, and to learn the protocols necessary to successfully 

accomplish the activity. Estimated breakdown for this travel expense: (2016 Domestic Per Diem Rates): 

Plane ticket ($400 x 2 = $800), lodging ($172/night x 2 nights = $314 x 2 = $688), meals per Diem ($69 x 3

days = $138 x 2 = $276), local travel ($200), other unexpected expenses ($200) = total $2,302. During the 

second year, the team will visit Dr. Allison O’Brien (Uniformed Services University, MD) to get acquainted 

with the techniques involved in Aim 1C of the activity, and to learn the protocols necessary to successfully 

accomplish the activity. Estimated breakdown for this travel expense: (2016 Domestic Per Diem Rates): 

Plane ticket ($200 x 2 = $400), lodging ($226/night x 2 nights = $452 x 2 = $904), meals per Diem ($69 x 3

days = $138 x 2 = $414), local travel ($200), other unexpected expenses ($200) = total $2,118. 

There will be some local travel costs between John Jay College, Hunter College, City College, and 

Albert Einstein College of Medicine. The amount requested for local MTA travel is $300. It is anticipated 

that some funds will be available from the college. Undergraduate student travel will be supported in part 

by the Program for Research Initiatives for Science and Math (PRISM), Collegiate Science and Technology 

Entry Program (CSTEP), and the Louis Stokes Alliance for Minority Participation (LSAMP). 

E. Participant/Trainee Support Costs: N/A

F. Other Direct Costs

Materials and Supplies: The cost estimate is based on the costs incurred during the PI’s previous award 

experiences, taking price increases into consideration, and a small amount that will be available from the 

department. PI requests $24,000 per year in supplies, reagents, and small equipment below $5,000. The 

supplies will include media for bacterial growth (Fisher Sci.: Tryptone, #BP1421-2, $660.14; Pepton, 

#BP1420-2, $487.47; NaCl, #S271-1, $80.71; Agar, #A360-500, $353.93; Sigma-Aldrich: Agarose, A9539-

250G, $608.00), plasmid, RNA, and protein purification media kits (Promega: DNA Clean-up wizard,

#A9282, $450.00; RNA purification kits, #AS1050, $359.00; Quagen: Plasmid purification kits, #10043, 

$276.00; DNA extraction kits, #28704, $113.00) and supplies, competent cells (ThermoFisher, Sci.: DH5α, 

#1825012, $212.00; BL21, C600003, $288.00; Sigma-Aldrich: Vero cells, V0180000, $205.50), restriction 

enzymes (New England Biolabs: NdeI, #R0111L, $249.00; XmaI, #R0180M, $262.00; StuI, #R0187M, 

$241.00; DNA and RNA ladders, buffers: N3232L, $208.00; N3231L, $220.00; N0364S, $65.00; #N0362S, 

$65.00; mutagenesis kit, #E0554S, $185 x 3), antibiotics (Fisher Sci.: Ampicillin, #BP1760-25, $103.76;

Chloramphenicol, #BP904-100, $202.45; Kanamycin, #BP2643-1, $101.97), RiboMax Transcription kits

and Rabbit Reticulocyte and Wheat Germ Lysates (Promega: SP6, P1280, $328.00; T7, P1300, $328.00; 

#L4960, $188.00; #L4380, $188.00), protein-protein interaction (ThermoFisher: #82015, $100.00) and 

protein labeling and cross-linking kits (ThermoFisher Sci.: #A10239, $427.00; #P6305, $306.00; #53031, 

$298.00), general biochemicals and solvents (Promega: Nuclease-free water, #P1197, $77.00; SigmaAldrich: DEPC, #D5758-100ml, $437.00; Fisher Sci.: ethanol, #BP8202-4, $248.25; n-butanol, #BP505-

500, $195.47; isopropanol, #A416-4, $208.89; methanol, #A456-4, $168.83; Across Organics: diethal ether, 

AC32686-0010, $78.28), reagents (Fisher Sci.: EDTA, #S312-500, $370.97 and #BP188-500, $105.95;

Contact PD/PI: Domashevskiy, Artem

Budget Justification Attachment Page 24
DTT, #BP172-25, $536.47; IPTG, #FERR0393, $540.00; protease inhibitor cocktails, PI-78415, $265.55;

Sigma-Aldrich: chloroacetaldehyde, #317276-250ml, $41.60; aniline, 242284-500ml, $82.00; TEMED, 

T7024-50ml, $63.50; (NH4)2S2O8, A3678-100G, $42.40; MP Biomedicals: PMSF, #ICN19538105, $90.22), 

and buffers (Fisher Sci.: Tris base, T393-500, $209.89; Tris-HCl, PR-H5123, $140.20; HEPES, 

ICN10559383, $232.20; MOPS, AC172635000, $301.88; MES, AC327765000, $465.99), electrophoresis 

supplies (Sigma-Aldrich: Acrylamide, A9099-500G, $188.00; bis-Acrylamide, 146072-500G, $97.60; TAE 

10x, #574797-1L, $40.20; TBE 10x, 574795-1L, $50.50; SDS, #L3771-500G, 229.00; BioRad buffers, gels, 

protein standards: #1610374, $125.00 x 2; #1610394S, $24.00; #1610373, $97.00; #1610375S, $135.00; 

#4561043EDU, $84.00 x 5; #4561045EDU, $84.00 x 5; #1610145EDU, $108.00; ), fluorescence and UVVIS cuvettes (Starna: #9/9-SOG-10, $98.00 x 4; #9B9-SOG-10, $129.00 x 4; #71-Q-10, $411.00; #9F-Q10, $252.00 x 4; #46F-Q-10, $520.00), pH electrode and buffers (Fisher Sci.: SB115-500, $43.56; SB99-500, 

$123.34; SB107-500, $41.86; #13-620-133, $491.80; #13-AB1-50AP, $660.25), chromatographic supplies

(GE Healthcare: HiTrap CM- #17-5155-01, $309.06; DEAE- #17-5154-01, $309.00, heparin- #17-0406-

03, $801.00; and Ni-Sepharose, #17-3712-06, $728.00 Fast Flow columns, ThermoFisher Sci.: C18 HPLC 

columns, #079013, $109.66 x 2; HPLC solvents), crystallography screens (Hampton Research, # HR2-142, 

$116.00; #HR2-134, $169.95; #HR2-084, $340.00; #HR2-086, $189.00; #HR2-107, $340.00; #HR2-109, $ 

340.00; #HR2-130, $189.00), Mettler Toledo balances (Fisher Sci. # 01-910-216, $4,690.00); UV 

Transilluminator (Fisher Sci. #11-993-16, $1,803.00); Microbiological incubator (Fisher Sci. #15-103-0515, 

$2,675.00); autoclaving tape and supplies, glassware, miscellaneous chemicals and crystallography reagents 

and supplies, NMR solvents, crystallography facility fees at City College, NMR facility fees at Hunter 

College, plasmid sequencing fees.

G. Direct Costs (Total): $297,254.27

H. Indirect Costs: The Research Foundation of CUNYo/b/o John Jay College of Criminal Justice has a 

federally-negotiated indirect cost rate agreement with the Department of Health and Human Services, at a 

rate of 69.5% of personnel including salaries and fringe benefits. Total requested is $148,131.10.

I. Total Costs: $445,385.37

Contact PD/PI: Domashevskiy, Artem

Budget Justification Attachment Page 25
Contact PD/PI: Domashevskiy, Artem

Tracking Number: GRANT12199590 Funding Opportunity Number: PA-16-200 . Received Date:

2016-06-22T16:33:35.000-04:00

RESEARCH & RELATED BUDGET - Cumulative Budget

Totals ($)

Section A, Senior/Key Person

Section B, Other Personnel

Total Number Other Personnel 5

Total Salary, Wages and Fringe Benefits

(A+B)

Section C, Equipment

Section D, Travel

1. Domestic

2. Foreign

Section E, Participant/Trainee Support

Costs

1. Tuition/Fees/Health Insurance

2. Stipends

3. Travel

4. Subsistence

5. Other

6. Number of Participants/Trainees

Section F, Other Direct Costs

1. Materials and Supplies

2. Publication Costs

3. Consultant Services

4. ADP/Computer Services

5. Subawards/Consortium/Contractual

Costs

6. Equipment or Facility Rental/User

Fees

7. Alterations and Renovations

8. Other 1

9. Other 2

10. Other 3

Section G, Direct Costs

(A thru F)

Section H, Indirect Costs

Section I, Total Direct and Indirect Costs

(G + H)

Section J, Fee

 Page 26
PHS 398 Cover Page Supplement

OMB Number: 0925-0001

Expiration Date: 10/31/2018

Tracking Number: GRANT12199590 Funding Opportunity Number: PA-16-200 . Received Date:

2016-06-22T16:33:35.000-04:00

1. Human Subjects Section

Clinical Trial? ❍ Yes ● No

*Agency-Defined Phase III Clinical Trial? ❍ Yes ❍ No

2. Vertebrate Animals Section

Are vertebrate animals euthanized? ❍ Yes ● No

If "Yes" to euthanasia

Is the method consistent with American Veterinary Medical Association (AVMA) guidelines?

❍ Yes ❍ No

If "No" to AVMA guidelines, describe method and proved scientific justification

3. *Program Income Section

*Is program income anticipated during the periods for which the grant support is requested?

❍ Yes ● No

If you checked "yes" above (indicating that program income is anticipated), then use the format below to reflect the amount and

source(s). Otherwise, leave this section blank.

*Budget Period *Anticipated Amount ($) *Source(s)

Contact PD/PI: Domashevskiy, Artem

 Page 27
PHS 398 Cover Page Supplement

Tracking Number: GRANT12199590 Funding Opportunity Number: PA-16-200 . Received Date:

2016-06-22T16:33:35.000-04:00

4. Human Embryonic Stem Cells Section

*Does the proposed project involve human embryonic stem cells? ❍ Yes ● No

If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell line(s) from the

following list: http://grants.nih.gov/stem_cells/registry/current.htm. Or, if a specific stem cell line cannot be referenced at this time,

please check the box indicating that one from the registry will be used:

Specific stem cell line cannot be referenced at this time. One from the registry will be used.

Cell Line(s) (Example: 0004):

5. Inventions and Patents Section (RENEWAL)

*Inventions and Patents: ❍ Yes ❍ No

If the answer is "Yes" then please answer the following:

*Previously Reported: ❍ Yes ❍ No

6. Change of Investigator / Change of Institution Section

❏ Change of Project Director / Principal Investigator

Name of former Project Director / Principal Investigator

Prefix:

*First Name:

Middle Name:

*Last Name:

Suffix:

❏ Change of Grantee Institution

*Name of former institution:

Contact PD/PI: Domashevskiy, Artem

 Page 28
 

PHS 398 Research Plan

OMB Number: 0925-0001

Expiration Date: 10/31/2018

 

Tracking Number: GRANT12199590 Funding Opportunity Number: PA-16-200. Received Date:

2016-06-22T16:33:35.000-04:00

Introduction

1. Introduction to Application

(Resubmission and Revision)

1242-Introduction.pdf

Research Plan Section

2. Specific Aims 1243-Specific_Aims_ADomashevskiy.pdf

3. Research Strategy* 1244-Research_Strategy_ADomashevskiy.pdf

4. Progress Report Publication List

Human Subjects Section

5. Protection of Human Subjects

6. Data Safety Monitoring Plan

7. Inclusion of Women and Minorities

8. Inclusion of Children

Other Research Plan Section

9. Vertebrate Animals

10. Select Agent Research

11. Multiple PD/PI Leadership Plan

12. Consortium/Contractual Arrangements  

13. Letters of Support 1245-Letters_of_Support.pdf

14. Resource Sharing Plan(s)

15. Authentication of Key Biological and/or

Chemical Resources 1246-Authentication_of_Key_Resources.pdf

Appendix

16. Appendix

Contact PD/PI: Domashevskiy, Artem

 Page 29
This is a resubmission (A1) of application 1R15AI124044-01, “Development of a Novel Inhibitor of Ricin: 

A Potential Therapeutic Lead against Deadly Shiga and Related Toxins,” which was reviewed in 

October 2015 at the MSFB study section. The initial submission received an impact score of 38. The initial 

submission was not funded. As a new investigator, I am grateful for the opportunity to present this revised 

application. I have made every effort to address the critique thoroughly, and I believe that the proposed studies 

have emerged considerably stronger and more focused on relevant aspects of structural quantification of RTAVPg interactions. This resubmission has undergone substantial revision, both in response to the reviewers’ 

comments and in order to meet the new guidelines for R15 application. For that reason, changes are not 

marked in the text.

From the summary statement for the A0 application, I understand that the reviewers thought “the overall impact 

of the application was moderately high.” The significance of the proposed research was deemed as “quite high,” and 

that the PI is “fully capable of carrying out the project.” The proposed research was thought to be “well-written, with 

clear rationale, and easy for undergraduate students to get involved.” The reviewers believed that “inclusion of well-planned 

education program was another major strength of the proposal, which provides resources and personnel to support training and 

supervision of these students.” However, the A0 application lacked preliminary data for RTA-VPg interactions and 

required biophysical methods of analysis, such as X-ray crystallography, and/or NMR spectroscopy, to fully

characterize and map-out RTA-VPg binding site. Similarly, the reviewers thought that molecular dynamics

simulation was not thoroughly though out. 

In this A1 application, we provide preliminary evidence for the RTA-VPg interactions (48.5 nanomolar 

affinity), as determined by fluorescence spectroscopy; this binding data were confirmed by performing 

multiples of titrations, yielding a standard deviation of 4.1. In addition, Scatchard analysis has revealed that 

there is one binding site on RTA for VPg. Furthermore, increasing concentrations of VPg inhibit the RTA 

N-glycosidase depurinating activity exerted on eukaryotic 28S rRNA in vitro, and we have quantified the amount 

of adenines cleaved by RTA in the absence and presence of VPg (appropriate controls were considered). The 

structure of RTA has been solved in the presence of tetranucleotide inhibitor (PDB ID 3HIO), and RTA has 

been crystallized previously under numerous conditions. Our preliminary data and the availability of RTA 

structure, provides us with a starting point for crystallizing RTA-VPg complex. First, we will characterize RTAVPg interactions thermodynamically and kinetically by employing fluorescence spectroscopy and isothermal 

calorimetry, and determine the minimum of VPg peptide that inhibits RTA activity in vitro and in vivo. Then, we 

will use two complementary structural techniques (crystallography and NMR spectroscopy) to map-out RTAVPg binding site. Creation of truncated VPg mutants in silico and application of docking algorithms will provide 

confirmation of our findings. To increase effectiveness, we will use tools of molecular modeling and site 

directed mutagenesis to create point mutations in VPg, further enhancing the binding and inhibition of RTA. 

We have initiated collaborations with Dr. David Jeruzalmi (X-ray crystallographer at Structural Biology Center, 

City College, CUNY), Dr. Nancy Greenbaum (NMR specialist at Hunter College, CUNY), and Drs. Shaneen 

Singh and Emillio Gallicchio (experts in molecular modeling, docking and scoring algorithms from Brooklyn 

College, CUNY), who have reviewed the corresponding aims for soundness, and have agreed to provide their

expert advice. 

I have addressed other concerns expressed by the reviewers as well. The team will visit collaborators’ 

laboratories to increase student involvement and study experimental techniques and procedures involved in 

off-site research. A section regarding biohazards was included. Isothermal calorimetry will be used as a 

complementary technique, and the collaborators have similar types of equipment to conduct parallel studies 

off-site. Both X-ray crystallography and NMR spectroscopy will be used, in parallel to molecular docking, to 

decipher and map-out RTA-VPg binding site. Molecular modeling was advanced and modified. An argument 

justifying the effectiveness of ricin toxin as weaponry was included. The conditions for achieving a stable RTAVPg complex were described and elaborated on. The physiological relevance of RTA-VPg complex and 

effectiveness of pharmacokinetic properties of peptide inhibitors were addressed. 

Thank you for your consideration. 

Contact PD/PI: Domashevskiy, Artem

Introduction to Application Page 30
Development of a Novel Inhibitor of Ricin:

A Potential Therapeutic Lead against Deadly Shiga and Related Toxins

Specific Aims

Ricin is a plant derived protein toxin produced by the castor bean plant. The plant is widely distributed 

throughout the world, and the toxin is well-known for its nefarious past. Global cultivation of the plant, and 

the ease of the toxin extraction from its beans, makes ricin a particularly appealing agent with a strong potential 

to be employed as a biological weapon. Ricin has been used as a homicidal poison since antiquity, having a long 

history of uses in espionage and warfare. In the past several decades, ricin has been associated with terrorist 

organizations in the US and other countries. The Centers for Disease Control (CDC) classifies ricin as a 

Category B biological agent. Currently, there is neither an effective vaccine that can be used to protect 

against ricin exposure nor a therapeutic to reverse the effects once exposed.

Ricin is a type 2 ribosome inactivating protein (RIP). RIPs are produced by many plants, effectively 

protecting plants against viruses, bacteria, and other parasites. RIPs are also dispersed among fungi, alga, and 

bacteria. The deadliness of RIPs has been explored by political and military organizations to design biological 

weaponry, scientists to generate transgenic species of plants resistant to viral and fungal infections, numerous 

cancer researchers in production of immune-conjugate therapeutics, as well as mystery writers to engage their

readers. RIPs are RNA N-glycosidases that inhibit advanced stages of protein synthesis by selectively modifying 

large rRNA molecules and deactivating ribosomes. 

While the majority of RIPs have thus far been isolated from plant species, the Shiga and Shiga-like 

toxins are produced by gram-negative bacteria species. Stx (Shiga toxins) are type 2 RIPs; they are produced by 

pathogenic STEC (Stx-producing E. coli) strains and are responsible for severe human illnesses, including 

hemorrhagic colitis, infantile diarrhea and hemolytic uraemic syndrome. RTA (ricin A-chain) and Stx1 bind to 

their target cell via a common receptor. X-ray crystal structures of RIPs, such as ricin and Shiga toxin, have 

been solved and reveal great similarities in their active sites. 

 We have identified that RIP activity of RTA is inhibited by a viral peptide associated with the turnip 

potyviral genome (VPg). VPg has been implicated as a link to a variety of viral functions, including overcoming

viral resistance in plants. High affinity of VPg for RTA, its ability to inhibit RTA enzymatic activity, and structural 

similarities in RIP active sites provide a new direction in the search for a novel generation of RIP inhibitors. Additionally, we 

have shown that a deletion mutant of VPg (VPg-71) is as effective an inhibitor as the full length VPg. The goal 

of the proposed research is to develop sufficient preliminary data to prove that VPg peptides can be used as 

RIP inhibitors. This knowledge will provide researchers with the tools to develop novel and more effective 

treatments against RIP toxicity, and advance strategies against the use of RIPs as agents of bioterrorism. In 

order to accomplish this goal, we propose the following two Specific Aims: 

Specific Aim 1: Establish conditions for RTA-VPg complex formation, and determine the minimum 

VPg peptide that binds and inhibits RTA. Based on structural similarities in RTA and Shiga toxin active 

sites, and our preliminary studies showing that VPg binds RTA with high affinity and inhibits its activity in vitro, 

we will determine how RTA-VPg interactions are prompted by administering direct fluorescence titrations and 

using either intrinsic or extrinsic protein fluorescence. Employing quantitative methods of analysis, we expect

to determine the extent of RTA inhibition by the VPg constructs, and identify thermodynamic and kinetic 

parameters of RTA-VPg interactions. 

Specific Aim 2: Mapping-out RTA-VPg interactions. Mapping-out of RTA-VPg binding is an important 

step in understanding how these proteins interact. Initial studies will use deletion mutants of VPg. Once we 

have identified the minimum VPg that binds and inhibits RTA, we will co-crystallize RTA-VPg complex, and 

use two complementary high resolution structural biology techniques (X-ray crystallography and NMR 

spectroscopy) to map-out RTA-VPg binding site residues. Creation of the mutants in silico and application of 

docking algorithms will verify our findings. To further enhance effectiveness, we will use tools for molecular 

modeling and site directed mutagenesis to create point mutations in VPg, further enhancing the binding and 

inhibition of RTA. 

Based on the outcomes of the Specific Aims above, we will test our inhibitor for the Shiga toxin in vivo. 

Contact PD/PI: Domashevskiy, Artem

Specific Aims Page 31
Development of a Novel Inhibitor of Ricin: 

A Potential Therapeutic Lead against Deadly Shiga and Related Toxins 

Research Strategy 

1. Significance 

 Ricin is a plant derived protein toxin produced by the castor bean plant (Ricinus communis L.). The toxin is 

well-known for its nefarious past. The plant is widely distributed throughout the world. Global cultivation of the 

plant and the ease of the toxin extraction from its beans made ricin particularly appealing agent with a strong 

potential to be employed as a biological weapon [1,2]. Ricin toxin (RT) has been used as a homicidal poison since 

antiquity [3]. The Centers for Disease Control (CDC) has classified ricin as a Category B biological agent [4]. 

Currently, there is neither an effective vaccine that can be used to protect against ricin exposure nor a 

therapeutic to reverse the effects once exposed.

 Ricin is a ribosome inactivating protein (RIP). RIPs are produced by many varieties of plants, effectively 

protecting members of the Plantae kingdom against viruses, bacteria, and other parasites [5-7]. RIPs are also 

dispersed among fungi, alga, and bacteria [8-11]. Plant RIPs are phytotoxins and deactivate ribosomes [12]. 

Examples of plant-derived RIPs include ricin, abrin (from Abrus precatorius), saporin (from Saponaria officinalis) and 

pokeweed antiviral protein (PAP) (from Phytolacca americana) [13-15]. Since their discovery, RIPs have been of 

great scientific interest due to their importance in human heath (as both pathogenic agents and therapeutics), and 

their potential uses in biological warfare and bioterrorism [16-21]. 

 RIPs are categorized into two major classes based on their physical properties. Type 1 RIPs consist 

exclusively of a single RNA N-glycosidase domain (e.g., PAP and saporin); they inhibit cell-free protein synthesis 

and are only mildly toxic to cells and animals. Type 2 RIPs (e.g., ricin, abrin, Shiga toxin, etc.), consist of an 

amino-terminal domain equivalent to type 1 RIPs (enzymatic domain with RIP activity; A-chain), joined by a 

disulfide bond to a different carboxyl-terminal domain with lectin properties (carbohydrate binding domain; Bchain) [22,23]. Type 2 RIPs are acutely toxic heterodimeric proteins. The lectin chain binds to galactosyl moieties 

of glycoproteins and/or glycolipids on the exterior of eukaryotic cells [24-26], promoting reverse transport of the 

A-chain to the cytosol [27-29]. The A-chain accesses translational machinery and depurinates ribosomes after it 

enters the cytosol. The extracellular location prevents contact between RIP and ribosomes of healthy cells, yet 

provides a direct source of toxin when the pathogen vector disrupts the cell. 

Reports of the use of ricin as a weapon date back to ancient times; ricin has a long history of use in 

espionage and warfare. Nonetheless, usage of this toxin as customary weapons is quite a modern idea. The ease 

by which one may acquire large amounts of ricin makes this toxin a prime candidate for bioterrorism. In 1978, 

ricin was employed in the assassinations of Markov [3,30]; many more recent instances were identified where this 

assassination technique was used [31]. In the past decade ricin has been associated with terrorist organizations in 

several countries [32]. The potential use of ricin toxin as a biological weapon of mass destruction has been 

highlighted in a FBI report released in 2007, entitled Terrorism 2002-2005, stating that “Ricin and the bacterial 

agent anthrax are emerging as the most prevalent agents involved in WMD investigations” [33]. 

Recent economic surveys show that there is over 1 million tons of castor beans produced in the world 

annually, having the potential to yield approximately 50,000 tons of pure ricin. The fate of much of this ricin in 

countries outside of the US is unknown. Ricin toxin can contribute up to 5% of the total protein of the bean 

(consumption of 8 beans is considered to be dangerous; subcutaneous toxicity is 24 μg/100 hours); it can be 

easily extracted from the mash produced as a by-product of castor oil production by several simple enrichment 

steps, and is therefore easy to accrue. In 1952 the US Army filed a patent on how to prepare ricin for weaponry 

purposes [34]. Before the 1990 Gulf war, the Iraqi military attempted to devise ways to disseminate ricin as an 

explosive bomb, attempts which were stalled by the outbreak of war and ensuing events. While it is true that 

ricin, and similar toxins are often cited as major bioterrorism threats, it is also understood that they are not very 

effective with respect to mass dispersal. It should be stressed that in recent years, bioterrorist mass attacks have 

given way to a “lone wolf” strategy, where the assault is directed by a single individual or small group, increasing 

the chances of fatalities. 

The majority of RIPs have thus far been isolated from plant species; although Shiga and Shiga-like toxins 

are produced by gram-negative bacteria species. Stx (Shiga toxins) [20,35] are produced by pathogenic STEC 

(Stx-producing E. coli) strains, which are responsible for severe human illnesses: including hemorrhagic colitis, 

Contact PD/PI: Domashevskiy, Artem

Research Strategy Page 32
infantile diarrhea and HUS (hemolytic uraemic syndrome) [36]. HUS is the main cause of acute renal failure in 

children and is the consequence of intestinal infection by STEC. 

In 2013 and 2014, the CDC reported a multistate outbreak of Shiga toxin-producing E. coli O121 (STEC 

O121), in which a total of 19 people were infected in the western United States; the infection was linked to raw 

clover sprouts and farm rich brand frozen food products [37,38]. Similarly, in 2014, the eastern US suffered 

another multistate outbreak of O157:H7 strain of E. coli; this specific outbreak was linked to ground beef 

contamination. Clearly, E. coli is still an important cause of human illness in the United States [39]. Among the 

multiple serotypes associated with disease, O157:H7 is responsible for more than 63,000 of the 175,000 total 

estimated STEC cases each year [40]. Ruminants, especially cattle, are the natural carriers of STEC, and these 

bacteria most commonly enter the food chain during beef processing [41-44]. Outbreaks and sporadic cases of 

STEC infection have also been attributed to contaminated fresh produce, person-to-person spread, and 

environmental sources [44,45]. Stx is the primary virulence factor associated with diseases caused by STEC. 

STEC produce two main types of bipartite toxins, Stx1 and Stx2, which are capable of binding to glycolipid 

receptors present on the surface of target cells through their B subunits [46]. After endocytosis, the A subunit 

damages ribosomes by releasing a specific adenine from 28S rRNA, and DNA, by releasing multiple adenines. 

The cytotoxic effects of RIPs have long been attributed to translation inhibition [47]. RIPs specifically 

target and depurinate the conserved sarcin/ricin loop (SRL) of the large rRNA [48]. The SRL within the rRNA 

of different ribosomes is universally conserved across all species [49]. Consequently, both the specificity of 

different RIPs for their substrates, and the selectivity between ribosomes among species, are likely to come from 

deviations within RIPs themselves and the ribosomal proteins. Ricin, for instance, presents the highest activity 

toward yeast and mammalian ribosomes, but exhibits low activity on plant and E. coli ribosomes, whereas PAP 

depurinates ribosomes from plants, bacteria, yeast, lower and higher animals, as well as a variety of viral RNAs 

[5]. RIP substrate specificity may be influenced by the deviations in ribosomal proteins, which dictate RIPs 

activity and sensitivity toward different ribosomes. The L9 and L10e rat liver ribosomal proteins are targeted by 

the ricin A-chain (RTA) [50], however PAP has been shown to bind to the L3 yeast ribosomal protein [51]. The 

structures of ricin, PAP and other RIPs have been solved, and the presence of various substrate analogs

interacting with RTA side chain amino acids has been mapped out [52,53]. RTA and PAP reveal a high degree of 

similarity in their active site residues (Figure 1A) [54-56]. Similarly, RTA and Stx1 bind to their target cell via a common 

receptor, in addition to having analogous amino acid residue distribution within their active sites (Figures 1B and 1C) [7,57,58]. 

Figure 1. (A) Superimposition of PAP and RTA [54] active site residues. RTA atoms are in thin lines; PAP is in thick lines [55]. 

In RTA, the substrate is sandwiched between Tyr123 and Tyr80, making six H-bonds to the Val81, Gly121 and Arg180 within its 

active site [56]. Structures of RTA and Siga toxin, Stx2 [7]: (B) Structure of RTA in complex with the cyclic tetranucleotide 

inhibitor; PDB ID 3HIO; (C) Structure of Shiga-like Stx2 A-chain from Escherichia coli in complex with adenine; PDB ID 2GA4. 

Preliminary Data: 

Viral protein (VPg) from turnip mosaic virus (TuMV) binds to and inhibits RTA activity in vitro. We 

have shown that PAP is inhibited by a viral peptide associated with the turnip potyviral genome (VPg) [59]. By 

employing fluorescence spectroscopy, we have determined that VPg is also able to bind to RTA with great 

affinity (Kd = 48.5 ± 4.1 nM) in a 1 to 1 ratio, and inhibit RTA depurination of eukaryotic 28S ribosomal RNA in 

vitro. Figure 2 presents the binding curve for RTA-VPg interactions, and the inhibitory effect of VPg on 

ribosomal depurination. 

A) B) C)

Contact PD/PI: Domashevskiy, Artem

Research Strategy Page 33
Figure 2. (A) Normalized fluorescence binding curve for a binary complex of RTA with VPg from TuMV. The inset depicts the 

Scatchard analysis at 25 oC of the titration data, indicating that there is one binding site on RTA for VPg. (B) Increasing 

concentrations of VPg inhibit RTA depurination of 28S rRNA in vitro, as determined by conversion of adenine released from 

rRNA to N6-ethenoadenine, and its quantification on the HPLC.

We have also shown that a deletion mutant of VPg (VPg-71; lacks 70 N-terminal amino acids) is as 

effective an inhibitor of PAP activity as full length VPg [59]. Our long-term goal is to understand how viral 

infections affect protein synthesis and cellular antiviral strategies. Turnip mosaic virus (TuMV) is a member of 

the genus Potyvirus that infects cruciferous plants (e.g., horseradish, cabbage, broccoli, cauliflower) throughout 

the world [60,61]. Several functions have been attributed to potyviral VPg, including viral replication, infection, 

and cell-to-cell movement; it has been shown to play an important role in the viral resistance of plants [15,59,62-

69]. We have proposed that VPg may provide an evolutionary advantage to the virus in the arms race with the 

plant defense mechanism – the RIP: VPg binds to PAP and inhibits its antiviral activity [15,59]. 

Ricin A chain (RTA) X-ray crystal structure has been solved. The structure of RTA has been solved and 

RTA has been crystallized under numerous conditions [56,70]. The 267 amino acid RTA consists of three 

domains: residues 1-117 contain a 6 stranded β-sheet that form the core of the protein and 2 α-helices; residues 

118-210 are comprised of 5 α-helices and contain active site residues, E177 and R180; and the region from 

residue 211 to residue 267 forms the interface with RTB and has a two stranded anti-parallel β-sheet and one αhelix [71]. Schramm laboratory have published the 2.0 Å X-ray crystal structure of RTA (Figure 1B), and this 

offers a starting point for crystallizing RTA-VPg complex, in that it was done with a tetranucleotide inhibitor. 

Scientific Premise: 

The high affinity of VPg for RTA, its ability to inhibit RTA enzymatic activity, and structural similarities in RIP active sites, 

provides a new direction in the search for a novel generation of RIP inhibitors and is the essence of the scientific premise of the 

proposed research. We seek to characterize RTA-VPg interactions, determine the minimum VPg necessary to 

efficiently inhibit ricin activity, and show that VPg peptides can be used as RIP inhibitors. This knowledge will 

provide researchers with the tools to develop novel and more effective treatments against RIP toxicity, allowing 

for strategies against the use of RIPs as agents of bioterrorism. Fluorescence spectroscopy and isothermal 

calorimetry (ITC) employed in this activity, are two complementary and commonly employed techniques that 

will allow our team to study protein-protein interactions [72,73]. To establish the stable conditions for RTA-VPg 

complex formations, we will vary pH, ionic strength, and buffer settings, leading to interactions that are enthalpically favorable and 

entropically driven, yielding the overall lowest energy. Transient protein complexes form and break rapidly, whereas 

permanent complexes have a relatively long half-life. Kinetic characterization of RTA-VPg will yield a full profile 

for these interactions; individual kinetics of RTA-VPg interactions (kon and koff) are very significant, and will 

dictate the biological activity of these proteins [74,75]. We have previously employed physiologically relevant 

translational assays and showed that PAP’s activity is inhibited in the presence of VPg [59]. Likewise, a rabbit 

reticulocyte lysate translational assay will provide physiological relevance to our experiments (in addition to in vivo

experiments performed by Dr. O’Brian). The RTA structure reveals that it is an alpha-beta protein with ABA 

sandwich architecture (PDB ID 3HIO) [76], and there is enough beta-strand character, yielding fine resolution 

spectra with sufficient dispersion to be assignable. First, however, a battery of 3D experiments needs to be 

A)

B)

Contact PD/PI: Domashevskiy, Artem

Research Strategy Page 34
performed on a 13C and 15N-enriched protein [77-80]. High yield of RTA expression is easily attainable from E. 

coli and/or yeast cells [81-84], and the protein remains clear in solution at concentrations suitable for NMR. 

 Currently, there is no inhibitor or therapeutic to counteract ricin poisoning, and those that have been 

investigated induce sever immune responses in animal models. This activity will provide the groundwork 

foundation and understanding into the mechanism of ricin inhibition by VPg peptide, providing models for 

designing molecules with greater inhibitory potency. In the early years of the pharmaceutical industry, and drug 

development pipelines, has been dominated by small molecules. For a drug to be marketed it has many obstacles 

to overcome; next to efficacy and tolerability, new drug candidates have to meet several requirements, including 

favorable pharmacodynamics (effects of a product related to its clinical activity), pharmacokinetics absorption, 

distribution, metabolism, and excretion), toxicity and safety issues [85]. Peptide attributes originally considered 

troublesome with respect to drug development may now turn out to be more convenient rather than unfavorable 

[85]. During the past decade, peptides have gained a wide range of applications in medicine and biotechnology, 

and therapeutic peptide research is also currently experiencing a renaissance for commercial reasons. Peptides are 

recognized for being highly selective and efficacious and, at the same time, relatively safe and well tolerated. 

Consequently, there is an increased interest in peptides in pharmaceutical research and development, and over 

140 peptides are currently being evaluated in clinical trials as therapeutics [86]. Rational design of peptide 

therapeutics can start with a known crystal structure of the peptide providing the secondary and tertiary 

structure. Then, based on the structure-activity relation, it is important to identify essential amino acids that are 

chemically prone to events such as isomerization, glycosylation, or oxidation, which should be avoided or 

substituted. Additionally, it is important to improve physicochemical properties of peptides, which often have 

the tendency to aggregate, sometimes having poor solubility. Strategies to avoid aggregation include the 

corruption of hydrophobic patches, which is achievable by means of substitution or N-methylation of particular 

amino acids. Changing the charge distribution and the isoelectric point of the peptide leads to increased 

solubility. The physicochemical properties of peptides can also be improved by the introduction of stabilizing αhelices, salt bridge formulations, or other chemical modifications, such as lactam bridges [86,87]. 

2. Innovation 

The PI proposes a high-impact experimental investigation of the effect exerted by a small genome-linked turnip 

mosaic virus protein (VPg) on the RIP depurinating activities of RTA and Shiga toxin. Ricin has been used in 

sporadic incidents of espionage and bioterrorism; therefore, there has been a great deal of interest in developing 

a safe vaccine or antidote to protect people from ricin poisoning. Although multiple types of vaccines have been 

tested and numerous ricin inhibitors or inactivators have been patented, none possess the ideal properties and 

standards of an effective vaccine. In terms of passive post-exposure protection, monoclonal neutralizing 

antibodies to passively protect animals are also under development. 

Many of the inhibitors developed and tested concentrate on structure-based designing of the inhibition 

of ricin’s N-glycosidase activity [56]. Recently, an inhibitor of the cellular trafficking of ricin has been reported 

[88]. Schramm et al. have reported oligonucleotide-based inhibitors of ricin [89]. The RiVaxTM (Soligenix) ricin 

toxin vaccine developed to protect against exposure to ricin toxin is the most advanced vaccine available to date 

[90,91]. The immunogen in RiVaxTM consists of a genetically inactivated subunit ricin A-chain that is 

enzymatically inactive and lacks residual toxicity of the holotoxin; this induces a protective immune response in 

animal models of ricin exposure, and functionally active antibodies in humans. 

Immunization after ricin exposure is impractical on a large scale; four hours after exposure to lethal 

doses the effects are irreversible, taking three to five days to kill an individual. Ricin is cleansed rapidly from the 

blood and the symptoms of ricin intoxication mimic those of other diseases. Furthermore, the current 

expectation behind vaccine development is that ricin would most likely be distributed as an aerosol. 

The research proposed by the PI and his team is innovative in nature; it involves investigating a novel 

peptide inhibitor of RIP activity of deadly ricin and Shiga toxin. Based on our experimental plan and preliminary 

data, we are confident that VPg constructs will serve as a new direction in the search for a novel generation of 

RIP inhibitors. Our research is an important milestone in an attempt to develop an effective vaccine for the 

inhibition of a wide variety of toxins with similar activities. A better ability to identify toxin in the body and 

improved vaccines [92] should mitigate the risk of its use in bioterrorism [3,93], in addition to providing more 

effective treatment for E. coli poisoning. 

Contact PD/PI: Domashevskiy, Artem

Research Strategy Page 35
3. Approach 

The PI and his research team (four undergraduate and two graduate students) propose a study that will 

investigate interactions between ricin A-chain (and Shiga toxin) and a viral peptide (VPg) from TuMV. We will 

determine the minimum VPg peptide required to efficiently inhibit RIP activity. The thermodynamic driving 

forces of these macro-molecular interactions are the critical parameters for the design of effective biomedical 

and pharmaceutical treatments. Once the minimum VPg peptide is established, we will crystallize the RTA-VPg 

complex and employ X-ray diffraction to map out the RTA-VPg binding site. Using bioinformatics tools, we will 

identify residue substitution in VPg, which may lead to more profound RIP inhibition. Our laboratory uses in 

vitro and in vivo analysis methods, and will investigate biochemical, molecular biological and biophysical aspects of 

this activity. This research is strongly collaborative in nature, combining the efforts of national and international 

institutions. Goals of this proposal will be accomplished by integrating scientific research knowledge, classroom experiences, 

collaborative efforts with other researchers, and an interdisciplinary research team involving underrepresented minority students. 

Materials and Methods 

VPg clones (VPg, VPg-71, VPg-81, VPg-91, etc.) are a gift from Dr. Miyoshi from St. Mariana University (Japan) 

and will be expressed in E. coli. We have a number of deletion mutants to test and since we have clones, other 

mutants can be readily created, both site specific and deletion. RTA clone is a gift from Dr. Schramm (Albert 

Einstein College of Medicine, NY); it is expressed in E. coli and purified in the PI’s laboratory at John Jay 

College. All protein purifications will be accomplished using an AKTAPure system (GE Healthcare). We have 

requested assistance from Dr. O’Brien (Uniformed Services University, MD), who is equipped to safely handle 

ricin and Shiga toxins. The PI and accompanying students will travel to the collaborators’ laboratories to learn 

the techniques used in this activity and assist in testing our inhibitor for that toxin as well. 

Bio- and Radio-Hazardous Materials and Safety: All personnel are required to undergo training in chemical 

waste disposal, and biochemical and radiological safety. A primer extension assay will involve the usage of a P-32 

radioisotope, an emitter of β radiation. All radiological samples and reactions will be performed in acrylic 

containers and tubes, with shields aiding in protection from radiation. Biohazardous and radiological chemical 

waste disposal will be carried out in accordance with the OSHA (Occupational Safety and Health 

Administration) regulations [94]. It should be noted that RTA cannot cross cell membranes and is not a 

bioterrorism agent in and of itself. The research requires no additional precautions for those experienced in 

working with PAP or RTA. Ricin and Shiga toxins are category B pathogens, and research involving these toxins 

will be performed in accordance with guidelines outlined by the OSHA by Dr. O’Brian’s laboratory. 

Rigorous Experimental Design

Fluorescence Data Acquisition and Analysis: Steady state fluorescence has been used extensively to study 

protein-protein and protein-nucleic acid interactions [95]. Fluorescence measurements will be taken at John Jay 

College in the PI’s laboratory using a Horiba Jobin Yvon FluoroMax®-3 (equipped with a 150 W xenon lamp, 

photodiode array detectors and FluorEssenceTM software). In experiments entailing high excitation intensity, a 

SPEX-Fluorolog-τ spectrophotometer (450 W) will be employed; the instrument is located in the Goss 

laboratory (Hunter College, CUNY). Fluorescence changes (quenching or enhancement) will be monitored using 

excitation and emission wavelengths appropriate to the experimental design. Data will be fit as previously 

described [96]. Briefly, data are normalized and subjected to statistical analysis using the following equation: 

 Fb = (Fobs – F0)/(((F∞ – Fobs)*(F∞/F0))+(Fobs – F0)) Eq.1 

where Fb is the fraction bound, Fobs is the observed fluorescence, F0 is the initial fluorescence value and F∞ is the 

final fluorescence value. Normalized values are plotted and analyzed by a nonlinear least-squares fitting for Kd

determinations. Data are fit and analyzed for both one- and two-site binding models. The uncertainty of fitting 

parameters, dilution corrections, and inner filter effects of titrant absorbance are considered in data analysis and 

interpretation. The software used is Graph Pad Prism®, version 5. 

Analysis of Stopped-Flow Kinetic Data: Kinetic experiments will be performed on an OLIS RSM 1000 

stopped-flow system (Dr. Dixie Goss, Hunter College) with a 1-ms dead time. Stopped-flow data for the binding 

of RTA to VPg will be analyzed using Global analysis software [97]. Data from the anisotropy experiments will be 

fitted to the single-exponential function: 

Contact PD/PI: Domashevskiy, Artem

Research Strategy Page 36
 rt = Δr exp (- kobs * t) + rf

 Eq. 2 

where rtis the observed anisotropy at any time t, and rfis the final value of anisotropy, Δr is the amplitude and kobs

is the observed first order rate constant; or the double-exponential function: 

 rt = Δr1exp (- kobs1 * t) + Δr2exp (- kobs2 * t) + rf Eq. 3 

where Δr1and Δr2 are the amplitudes for the first and second components of the reaction with observed rate 

constants kobs1 and kobs2, respectively. Data will be subjected to statistical analysis.

NMR Spectroscopy: Acquisition of 3D HNCO, HN(CA)CO, HNCA, HN(CO)CA, HNCACB, and 

CBCA(CO)NH spectra to assist in assignment of backbone resonances Cα, Cβ, CO, and amide N and 1H of the 

RTA protein [98], and 2D [1H, 15N]-HSQC spectra to monitor chemical shift changes upon binding of VPg, will 

be performed at 298 K on a 600 MHz Bruker Avance spectrometer (equipped with a three-channel TXIcryoprobe) in the NMR Facility at Hunter College with the aid of Dr. Nancy Greenbaum. NMR experiments will 

be acquired and processed using Bruker TopSpin 3.2 software in triplicates and statistically analyzed. Spectral 

visualization and chemical shift assignments will be performed with SPARKY [99]. For the experiments, samples 

of RTA will be labeled with 15N and/or 13C (as described in Aim 2) and will be carried out in in 90% H2O/10% 

D2O phosphate buffer in 5 mm micro volume NMR tubes (Shigemi, Inc.). Changes in 15N and 1H chemical 

shifts of the protein observed in a [1H-15N]-HSQC spectrum will be calculated using an adjusted chemical shift: 

 Δδ = [(ΔδH)

2 + (0.2 * ΔδN)

2

]

1/2 Eq. 4 

 

where ΔδH represents the chemical shifts change observed for amide 1H and ΔδN for amide 15N. The mean and 

standard deviation σ of the shifts will be calculated and most perturbed residues will be identified as those whose 

shift changes are greater than σ. The fit will be performed using the SOLVER function of Graph Pad Prism®, 

version 5, by minimizing the sum of squared errors for Δδ.

We plan to accomplish our goal by directing the following Specific Aims and Rigorous Experimental Design: 

Specific Aim 1: Establish conditions for RTA-VPg complex formation, and determine the minimum 

VPg peptide that binds and inhibits RTA. Based on structural similarities in PAP, RTA, and Shiga toxin 

active sites, and our preliminary studies showing that RTA binds to VPg with high affinity, and that increasing 

concentrations of VPg inhibit RTA activity in vitro, we will determine how RTA-VPg interactions are prompted 

by administering direct fluorescence titrations and using either intrinsic or extrinsic protein fluorescence. 

Employing quantitative methods of analysis, we expect to determine the extent of RTA inhibition by the VPg 

constructs, and identify thermodynamic and kinetic parameters of RTA-VPg interactions. 

Aim 1A: Characterize RTA-VPg interactions and identify minimum VPg peptide that binds to RTA. We

will purify recombinant RTA, wild type VPg, and VPg mutants, and measure the equilibrium constants for RTAVPg protein-protein interactions in a range of temperatures (e.g., 10, 20, 30 oC) using direct fluorescence 

titrations to determine pivotal thermodynamic binding parameters (e.g., ΔH, ΔS and ΔG) for RTA-VPg complex 

formation, and identify whether the binding is entropically favored or enthalpically driven. All titration 

experiments will be performed in triplicates and statistically analyzed to extract standard deviations. Once the 

basic binding parameters for wild type VPg-RTA complex are established, and the binding has been 

quantitatively characterized, we will determine the minimum VPg peptide required to bind to the RTA. All VPg 

constructs (VPg-71 through VPg-121 deletions and wild type VPg) are His-tagged, easing their purification by 

Ni-Sepharose affinity chromatography. We will measure binding constants for RTA-VPg constructs using direct 

fluorescence titrations and establish the affinity between the proteins. Those VPg mutants that do not bind RTA 

will not exhibit saturation binding behavior, nor will we observe the decrease in adenines released from the 

depurinated substrate RNA. The changes in fluorescence upon binding are directly related to the interaction of 

the two proteins. For intrinsic protein fluorescence (λex= 280nm/λem = 332nm), the difference between identical 

concentrations of RTA and VPg, taken separately and when mixed together, will give the difference in 

fluorescence for the complex. The fluorescence intensity of RTA will be measured, then blank buffer will be 

titrated in, adding to the corrected intensities (Fs). Once RTA is titrated with VPg, the corrected fluorescence for 

the complex will be obtained (Fc). The difference in fluorescence intensity related to the complex is defined as 

ΔF = Fc – Fs. The inner filter corrections will be applied, whenever necessary, using the equation: 

Contact PD/PI: Domashevskiy, Artem

Research Strategy Page 37
 Fcorr = Fobs· antilog [(Aex+Aem) / 2] Eq. 5 

where Fcorr and Fobs are the corrected and observed fluorescence intensities, respectively. The absorbance of the 

samples will be measured using an UV-VIS spectrophotometer. The normalized fluorescence difference 

(ΔF/ΔFmax), between the protein-protein complexes and the sum of the individual fluorescence spectra, will be 

used to determine the equilibrium dissociation constants (Kd) for the interactions at different temperatures, 

allowing for the construction of a van’t Hoff plot. We will vary pH, ionic strength, and buffer settings. 

Expected Outcomes and Alternative Approaches. At the conclusion of these experiments, we will 

quantitatively determine equilibrium binding constants for the interactions of RTA with VPg peptides, and will 

have detailed quantitative measurements of RTA-VPg protein-protein interactions. The energy profile of the 

RTA-VPg binding reaction is the final result of the combined fluorescence and ITC approach, and a cornerstone 

in the elucidation of these protein-protein interactions. The current deficiency in physical measurements will be 

addressed and our description of these interactions will lead to an understanding of these novel RTA-VPg 

interactions. Furthermore, the data will allow for comparisons of binding and VPg effects with other RIPs (e.g., 

PAP), providing comparative information of these interactions. Being the initial studies, they are particularly 

important. Several complementary and alternative approaches to the above Aim are described below.

Isothermal Titration Calorimetry (ITC): As a complementary approach to fluorescence, and to confirm the 

veracity of RTA-VPg thermodynamics, isothermal titration calorimetry (ITC) [73,100] will be employed. The PI 

and his students will travel to Denver University, and assist Dr. John Latham (Department of Chemistry and 

Biochemistry, Denver University, CO), in using the Nano ITC (TA Instruments) to determine the 

thermodynamics of RTA-VPg interactions in solution. Calorimetry is an excellent technique, allowing for 

characterization of the thermodynamic driving forces and defining molecular interactions. All binding events are 

accompanied by the evolution or absorption of heat (ΔH). With the appropriate experimental design, 

fundamental information regarding the molecular interactions driving the process, as well as stoichiometry of 

binding (n) and the binding constant (Ka), can be generated. The ITC experiment will be performed at a constant 

temperature by titrating one binding partner (VPg, the titrant) into a solution containing the other binding 

partner (RTA; the titrand) in the sample cell of the calorimeter. All experiments will be performed in triplicates.

After each addition the heat released or absorbed in the sample cell is measured with respect to a reference cell 

filled with a reference buffer sample. Information obtained from the ITC experiment will include free energy, 

enthalpy, entropy, heat capacity changes, and any protonation effects that accompany RTA-VPg binding 

interactions. ITC will provide the global thermodynamic parameters of the RTA-VPg binding reaction. Data 

from non-calorimetric methods will complement this characterization [101]. 

Kinetic characterization of RTA-VPg interactions: Protein motions, particularly their conformational 

dynamics, regulate and often constitute protein function. Probing the structural and internal dynamics of RTAVPg complexes requires complementary experiments, allowing energetic and dynamic aspects in the formation 

and stability of the complex to be revealed. Fluorescence anisotropy studies will provide information on the 

binding mechanisms, and may lead to mechanistic models of RTA inhibition on the molecular level. To gain 

insight into the kinetic mechanism of RTA-VPg binding, and understand how this binding is affected, we will 

perform stopped-flow anisotropy measurements for RTA binding to VPg [72]. All experiments will be 

performed in triplicates. We will label RTA (or VPg) with fluorescein (λex = 490 nm, λem = 520 nm) to aid in the 

sensitivity of measurements. The temperature of the flow-cell and solution reservoir will be maintained constant

at 25 oC. After rapid mixing of RTA with various concentrations of VPg, the time course of the anisotropy 

change will be recorded by computer data acquisition. Statistical analysis of the acquired will be performed. In 

each experiment, 1000 pairs of data will be recorded and sets of data from 5 to 7 shots will be averaged to 

improve the signal-to-noise ratio. Data will be evaluated by fitting to the single- and double-exponential 

functions [102,103]. Each averaged set of stopped-flow anisotropy data will then be fitted into nonlinear 

analytical equations using Global analysis software provided by OLIS. For the stop-flow fluorescence 

measurements, we will excite RTA and will measure the fluorescence (voltage) after passing a 324 nm cut-on 

filter. A reference photomultiplier will be used to monitor fluctuations in the lamp intensity. The temperature of 

the flow cell and solution reservoir will be maintained by using a temperature controlled circulating bath. 

Corrections will be made to the anisotropy changes when titrating a buffer blank into RTA. 

Contact PD/PI: Domashevskiy, Artem

Research Strategy Page 38
Extrinsic Fluorophore Labeling: Should the fluorescence difference spectra prove too small to give accurate 

results, as an alternative approach, we will label one, or both, of the proteins with an extrinsic fluorophore. There 

are wide varieties of probes to choose from (e.g., NHS-fluorescein from Pierce has λex = 493 nm and λem = 516 

nm that are far removed from native protein fluorescence). Typically, labeling is done at neutral pH so that only 

uncharged amines are labeled. Using a limited amount of probe, one or two sites are labeled on the protein. 

Determination of the amino labeled site will be confirmed by digestion of a control, unlabeled protein sample, 

and HPLC analysis. Binding of NHS-fluorescein-labeled RTA to VPg will serve as a functional comparison for 

the labeled and unlabeled proteins. We can label either protein, and should this method be necessary, labeling of 

each protein separately and titrating with the other will provide a control that the probe has not perturbed the 

equilibrium [59]. Simultaneous measurements of the individual protein spectra and of all buffer solutions will 

also be used as controls for the changing fluorescence signal. Data will be analyzed as described in Fluorescence 

Data Acquisition and Analysis. Averages of at least three titrations and use of at least two different protein 

preparations will be used as controls to verify the data. As an additional control, using catalytically inactive RTA, 

measurements will be made to determine whether it still binds to VPg, and if so, how it compares to native RTA. 

Aim 1B: Determine the extent of rRNA depurination by RTA in the presence and absence of VPg 

peptides, and identify shortest VPg necessary to inhibit RTA. After establishing favorable conditions for 

the minimum VPg peptide binding to RTA, we will assay the inhibition of ricin activity, and correlate binding to 

depurination. Analysis of ricin activity will be determined as described previously [59]. Briefly, the amount of 

adenine released during depurination of eukaryotic rRNA will be determined by conversion of the purine to its 

fluorescent derivative, N

6

-ethenoadenine, using chloroacetaldehyde. Samples will be analyzed with the Waters 

HPLC located in the PI’s laboratory. The HPLC is equipped with Waters 2487 dual λ absorbance detector (set at 

254 nm), Waters 2475 multi λ fluorescence detector (λex = 315 nm and λem = 415 nm), controller, and an 

autosampler. The column to be used is a reverse-phase XBridgeTM C18 column, and the elution solvent is 

ammonium acetate/methanol system. The areas under the elution peaks will be analyzed by Waters EmpowerTM

chromatography software, and related to the amount of adenines released from rRNA by RTA activity. The 

retention time of the elution peak and the internal standard, N

6

-ethenoadenine, will be used for the 

quantification of the adenines released by RTA. Eukaryotic 28S rRNA will provide a positive control for the 

experiments, whereas poly(C) will serve as a negative control for the depurination. Standard curve will be created 

and data will be statistically analyzed. We will then perform a primer extension assay on the RTA-depurinated 

rRNA substrate, determining and confirming the location of the depurination site. All of the above experiments 

and assays will be performed at least three times.

Expected Outcomes and Alternative Approaches. At the conclusion of these experiments, we will determine

to what extent rRNA is depurinated by RTA, in the absence and presence of VPg peptides, and correlate this 

depurination with the physical binding measurements (see Aim1A). We expect to have detailed quantitative 

measurements of the effects exerted by VPg on the activity of RTA. Several alternative and complementary approaches to 

the above methods are described below.

Aniline Cleavage Assay of N-glycosidic Activity: Alternatively, the depurination can be determined by an 

aniline cleavage assay of N-glycosidic activity. After the adenine is removed the depurinated site becomes 

unstable, and is subjected to a β-elimination reaction when RNA is treated with acidic aniline. This cleaves the 3’-

end of the RNA and the depurination product is detected by electrophoresis [104,105]. As a control, aniline 

treatment of RNA in the absence of RTA will confirm that the target RNA sample is free of ribonuclease 

activity. The extent of depurination is determined based upon the amount of nucleic acid chain cleavage 

[104,106]. The assays will be performed in triplicates.

Luminescent Assay for RTA Activity: In addition to the above methods to assay RTA activity, we will employ 

a sensitive luminescent coupled assay [107], for the measurement of adenine released from rabbit reticulocyte 

80S rRNA by RTA in the presence or absence of VPg protein. Adenine phosphoribosyl transferase (APRTase) 

and pyruvate orthophosphate dikinase (PPDK) convert adenine released by RTA to ATP for quantification by 

firefly luciferase. The resulting AMP is cycled to ATP, so it produces sustained luminescence proportional to the 

adenine concentration [107]. Continuous measurements are accomplished in 96-well plate format by combining 

the luciferase reagent with APRTase/PPDK (adenine to ATP) coupling enzymes. Both, APRTase and PPDK 

Contact PD/PI: Domashevskiy, Artem

Research Strategy Page 39
clone from Clostridium symbiosum are gifts from Dr. Schramm (Albert Einstein College of Medicine). Assay 

samples lacking VPg will constitute a positive control of the depurination, whereas RTA depurination assay 

samples lacking RIP will generate a negative control for this experiment. 

Aim 1C: Identify the effect exerted by VPg peptide on Shiga toxin activity. Depending on the outcomes of 

the Aims 1A and 1B, the PI has arranged to send out VPg peptides to his collaborator at the Uniformed Services 

University of the Health Sciences, Dr. O’Brien, to test the effect of the VPg inhibitor on Shiga toxin (Stx) 

toxicity on Vero cells (CCL-81) [46]. The cells will be transfected with cDNA encoding for VPg peptides, and a 

cytotoxicity assay will be performed by Dr. O’Brien, revealing any inhibitory effects of VPg on cytotoxic ricin 

and Shiga toxin. The PI will visit Dr. O’Brien’s laboratory to be fully involved in the techniques performed. As a 

negative control, a different His-tagged protein will be used; a positive control of ricin or Shiga toxin will lack 

VPg peptides. All experiments will be performed in triplicates.

Ricin and Shiga toxin (Stx) Cytotoxicity Assays: The Vero cytotoxicity assay [108,109]: Vero cells will be 

seeded in 96-well plates under appropriate conditions, and incubated in the presence and absence of Shiga toxin 

or intact ricin toxin. Consecutively, the inhibitory effect of VPg on the toxin will be tested by successive addition 

of the viral peptide to the toxin treated Vero cells. The cells will then be fixed with formalin and stained with 

crystal violet. The absorbance of the wells is then measured at 630 nm using a spectrophotometer. 

Expected Outcomes and Alternative Approaches. We expect to have a detailed quantitative data on the 

inhibitory effect of VPg on the cytotoxicity of Shiga toxin and ricin toxin. These expectations stem from our 

preliminary data, and a central hypothesis stating that VPg will successfully bind to and inhibit the RIP activity of 

deadly Shiga toxin. This is essential in developing novel vaccines for the prevention of E. coli poisoning. As an 

alternative approach to the Vero cell cytotoxicity assay, HCT-8 cell line will be used in the same manner as Vero 

cells, except that the cells must be incubated prior to the toxin application. Controls will be similar to the Vero 

cell assay. The CD50 will be determined by the reciprocal of the toxin dilution that caused 50% of the cells in a 

monolayer, compared to control cells. Specific toxin activity is calculated as CD50/mL divided by the toxin 

concentration in mg/mL [46]. 

Specific Aim 2: Mapping-out RTA-VPg interactions. A comprehensive understanding of RTA-VPg binding 

is an important step in understanding how these two proteins interact. Initial studies will use deletion mutants of 

VPg. Once we have identified the minimum VPg that binds and inhibits RTA, we will express these VPg 

mutants in either E. coli or purchase custom made truncated VPg peptides. We will co-crystallize RTA-VPg 

complex, and use two complementary high resolution structural biology techniques in parallel (X-ray 

crystallography and NMR spectroscopy) [110] to map-out RTA-VPg interacting residues. Creation of the 

mutants in silico and application of docking algorithms will confirm our findings. To further enhance 

effectiveness, we will use tools for molecular modeling and site directed mutagenesis to create point mutations in 

VPg, further enhancing the binding and inhibition of RTA. 

Aim 2A: X-ray crystallography to define sequence requirements of VPg binding to RTA. The structure of 

RTA has been solved and RTA has been crystallized under numerous conditions [70,76]; however, no structure 

exists of VPg or RTA-VPg complex. We will seek crystallization advice, X-ray diffraction, and data analysis from 

the PI’s collaborator Dr. David Jeruzalmi (City College, CUNY), who is an expert in this technique. To prepare 

crystals for structure determination using X-ray crystallography, we will initially follow a novel procedure for 

RTA-inhibitor crystallization established by the Schramm laboratory [76]. As necessary, crystallization trials for 

the entities under investigation here will also be carried out using the index, crystal and detergent screens 

(Hampton Research). These commercially available screens will allow us to sample 230 crystallization conditions; 

other screens available in the group of Dr. Jeruzalmi will also be tried. Once the ideal conditions for RTA 

crystallization are developed, we will soak RTA crystals with various concentrations of VPg (2-3-fold excess of 

RTA), and concentrate the drops by exposing them to dry air before mounting and freezing the crystals in liquid 

nitrogen. If the X-ray diffraction data acquired from these crystals yield weak electron density, we will vary 

crystallizing conditions to obtain crystals with higher VPg occupancy. X-ray diffraction data will be collected 

using Beamline X29A (CCNY) and the data will be processed with the HKL2000 program suite [111] and 

statistical software. The PI will incorporate the screening of VPg crystallization into the undergraduate 

biochemistry course offered by the Department of Sciences. Hampton Research provides crystallization screens 

Contact PD/PI: Domashevskiy, Artem

Research Strategy Page 40
that are optimally designed to provide rapid screening of the crystallization of macromolecules. This is a highly 

effective approach to overcome the exhaustive search for suitable crystallization conditions; the formulation 

utilized evaluates 96 unique mixtures of pH, salts, polymers and organics, and their ability to promote crystal 

growth. In the event of unsuccessful crystallization, students will still gain valuable experiences in protein 

crystallography. X-ray diffraction data will be collected from several acquired crystals and compared for accuracy 

and consistency.

Expected Outcomes and Alternative Approaches. At the conclusion of these experiments, we expect to have

a detailed description of interacting amino acid residues in RTA-VPg complex by probing the molecular basis of 

recognition between the proteins and employing X-ray crystallography. The technique requires fine crystals, and 

if we encounter difficulty in generating nicely diffractable crystals, we will send protein samples to HauptmanWoodward Medical Research Institute (University of Buffalo, NY), which provides crystallization services. 

NMR Spectroscopy: As an alternative to crystallography, we will employ NMR spectroscopy, a complementary 

technique used to map-out macromolecular interactions. NMR will require an isotopic enrichment with 13C and 

15N of one of the proteins, and then the other; this will aid with NMR chemical shift assignments. We expect to 

identify chemical shift changes observed upon titration of RTA with VPg peptide that inhibits RIP activity. The 

PI and his students will employ NMR studies to map out interactions between RTA and VPg, gaining insight 

into the amino acid interactions between the proteins and realizing the sequence requirements for binding. To 

identify interacting amino acid residues and map out the interface of the binding site, we will analyze 

perturbations in resonances corresponding to amide N and H of uniformly 15N-labeled RTA (or VPg as a 

control). The 1H-15N HSQC spectrum of RTA will allow us to assign sequence specific chemical shifts to the 

protein. Addition of stoichiometric amounts of VPg will result in the quantifiable perturbation of a number of 

backbone amide cross-peaks. Mapping the residues with the greatest chemical shift perturbations, in the presence 

and absence of VPg, will allow for the identification of these interacting residues. We will then fit the chemical 

shift perturbation data for each perturbed residue to an isotherm, calculated by the hyperbolic equation upon 

titration of 15N-enriched RTA, with increasing amounts of VPg to derive the apparent Kd for each perturbed 

residue. This constant will be compared to the one acquired in the Aim 1. For isotopic enrichment with 13C and 

15N, uniformly 13C/15N labeled recombinant RTA will be prepared to assist with NMR chemical shift 

assignments. The isotopically enriched protein will be obtained using a method developed by Bracken et al.,

adapted to RTA [112]. 

Aim 2B: Employ tools of molecular modeling/docking to map out RTA-VPg interactions. After we 

identify the RTA-VPg interface residues, the PI and his students will employ bioinformatics tools; molecular 

modeling, docking and scoring algorithms will map out molecular interactions between RTA and VPg proteins, 

gaining insight into the dynamics of amino acid interactions. We will seek advice of Dr. Shaneen Singh and 

Emilio Gallicchio (Brooklyn College, CUNY), experts in molecular modeling. Molecular docking allows 

prediction of the orientation of two molecules relative to each other. Approaches include: (i) using 

complementary in shapes, and (ii) simulation of binding. Small molecule structures will be obtained from 

DrugBank 3.0. We are able to perform these structure-based virtual screening exercises because of readily 

available X-ray crystallographic structures for RTA (PDB ID 3HIO) and Shiga toxin (PDB ID 2GA4) proteins. 

The search for the correct binding mode (pose prediction) of the VPg constructs will be carried out by 

performing a number of trials and keeping those energetically best. This involves finding the correct orientation, 

and as most protein molecules are flexible, the correct conformation of the docked molecule. In order to explore 

a large search space, we will explore algorithms, specifically developed to keep track of previously discovered 

minima and guide the search into new regions [113,114]. The team will then apply affinity scoring functions to 

the energetically best pose, or n best poses for each of the RTA-VPg complexes, comparing the affinity scores 

for different complexes, thus providing their relative ranking-order. The assumption is that for a given molecule 

the best pose according to the affinity score is among the n saved poses identified with the dock score [115,116]. 

A large number of docking programs and search algorithms have been published. One criterion for classifying 

the underlying algorithms is how the proteins are threaded during docking. To start, we will use a program that 

builds the protein incrementally, starting from a docked ‘base fragment’ – FlexX [117]. Similarly, we will use a 

program, such as Ligand Docking (GOLD) [118], to thread the protein sequence in its entirety. 

Contact PD/PI: Domashevskiy, Artem

Research Strategy Page 41
Expected Outcomes and Alternative Approaches. After the completion of this Aim, we will have a detailed 

description and identification of the amino acid residues that participate in the RTA-VPg binding interactions in 

silico. This will allow us to virtually substitute individual amino acids in the constructed models, and identify those 

substitutions that yield the lowest binding energy while retaining the overall structure and inhibitory 

characteristics. This will provide a future direction in the search for a novel peptide sequences that inhibit RIP 

activity of RTA and Shiga toxin RIPs. For those VPg deletion constructs whose structures are not available, we 

will resort to other techniques of protein structure prediction. As an alternative, we will apply ‘threading’ and 

homology modeling to determine these structures [119,120]. Hammerhead [121], AutoDock, and DOCK 

[122,123] programs may be used alternatively as docking algorithms. We will employ rational design of peptide 

therapeutics that will improve binding affinity of VPg for RTA or Shiga toxin and inhibition of their toxicities. 

Based on structure-activity relation, we will identify amino acids that promote isomerization, glycosylation, 

aggregation, and/or oxidation (Met, Cys, His, Trp, Tyr, etc.) and substitute them via site-directed mutagenesis 

(Aim 2C) to avoid these events [86,87,124].

Aim 2C: Site-directed mutagenesis of VPg to increase its inhibitory effect on RTA. In vitro site-directed 

mutagenesis is an invaluable technique for studying protein structure-function relationships and gene expression, 

and for carrying out vector modifications [125]. Several approaches to this technique have been described [126-

129]. These methods generally require single-stranded DNA (ssDNA) as the template and are labor intensive or 

technically difficult. We will use Stratagene’s QuickChange® Site-Directed Mutagenesis Kit that will allow sitespecific mutations to be performed in virtually any double-stranded plasmid, thus eliminating the need for 

subcloning into M13-based bacteriophage vectors. The system requires no specialized vectors, unique restriction 

sites, or multiple transformations. The process is rapid, generating mutants with greater than 80% efficiency. The 

positive control reaction for the mutagenesis will include pWhitescript 4.5-kb plasmid, which produces known 

mutations under the conditions of the reaction. 

Expected Outcomes and Alternative Approaches. At the conclusion, we will successfully create a series of 

VPg constructs (e.g., constructs with point mutations, amino acid switching, and/or deletion/insertion of single 

or multiple amino acids) which, as predicted by the tools of bioinformatics, would have a stronger binding 

affinity for RTA and more pronounced inhibitory effect on its RIP activity. We will then express these 

constructs, and purify to homogeneity for further binding and enzymatic studies and testing.

Summary 

Ribosome inactivating proteins (e.g., ricin and Shiga toxin) are highly toxic agents produced by the castor bean 

plant and pathogenic enteric gram-negative bacteria, respectively. Presently, there are no effective inhibitors of 

RIP activity. This proposal seeks to develop a novel peptide inhibitor of RIP toxicity, examine its interactions 

with RTA and Shiga toxin, and improve the potential to provide a therapeutic lead against these deadly toxins. 

This research is strongly collaborative in nature; it will provide excellent training opportunities for undergraduate 

and graduate students, in multiple biomedical and biophysical techniques, as well as data analysis. Additionally, 

by developing a vast network of partnerships among scientists from local, national and international institutions, 

this activity will enhance the infrastructure for research and education at John Jay College. 

Tentative Timeline of the Proposed Activity: We expect to begin our proposed experiments in the fall of 

2016, and anticipate for all studies outlined in this proposal, to be completed within 3 years, by the end of 2019 

to early spring of 2020. Year 1 (2016-2017) will be devoted to purification of recombinant RTA and VPg 

proteins, and execution of fluorescence and ITC experiments that will characterize thermodynamics of RTAVPg binding interactions (Aim 1A). We will also initiate crystallization studies during the first year (Aim 2A).

During year 2 (2018), RTA-VPg interactions will be kinetically characterized by stop-flow anisotropy and 

fluorescence (Aim 1A). We will also probe catalytic assay for RIP activity and investigate VPg inhibitory effects, 

employing depurination assays (Aim 1B). VPg peptides will be sent to Dr. O’Brien’s laboratory, who will examine 

inhibitory effects of VPg on ricin and Shiga toxins in vivo (Aim 1C). Crystallography of RTA-VPg complex, X-ray 

diffraction and NMR will begin during year 2 (Aim 2A) and continue into year 3 (2019). During year 3 (2019-

2020), RTA-VPg binding studies we will performed by implementing extrinsic fluorophore labeling. Molecular 

modeling, docking and scoring algorithms, as well as site-directed mutagenesis, will be performed during year 3. 

The team will visit collaborators’ laboratories, and get familiar with techniques and protocols described in this 

Contact PD/PI: Domashevskiy, Artem

Research Strategy Page 42
activity. The laboratory technician will be responsible for preparation of routine biological media, plasmid 

preparation, protein purification and assays, equipment maintenance, etc. All research assistants will participate 

extensively in this project. The PI’s central career goal is to establish a productive research program at John Jay 

College that engages students in modern experimental biomedical research techniques. In addition to enhancing 

the infrastructure of the College, this funding will be critical toward his career advancement, and will contribute 

to the maintenance of an on-going research program in an important area of plant defense mechanisms. We 

expect to generate enough data for at least one publication per each funded year and present our findings at the 

annual American Plant Biology Society and the Biophysical Society meetings. The funding will also foster 

networking and establish collaborations between the PI’s and fellow scientists. This research is the collaborative 

effort of the PI, his students, and professional colleagues, and is an important contribution to his on-going 

research. It will provide opportunities for guest lecture invitations at other research universities, serving on grant 

review panels or journal editorial boards, further advancing the PI’s career. The above activity will ensure the 

PI’s competition for other funding mechanisms, such as collaborative grants. As an NIH supported laboratory in 

the Department, AREA support will foster the introduction of biomedical research to students not currently 

exposed; this is an essential resource for minority students interested in pursuing a career in biomedical research. 

Mentoring Statement: The PI has developed his research career mentoring undergraduate and Master’s 

students in a vibrant, diverse urban university system. All of the proposed projects are available for 

undergraduate student participation. Student researchers working in the PI’s laboratory will have completed a 

minimum of their sophomore year and will co-enroll for the Undergraduate Research Internship course (FOS 

402); this course is a capstone experience for the Forensic Science Major with hands-on teaching and laboratory 

training. The course is intended for students interested in graduate school and research careers. Since fall of 

2015, the Department offers new majors in Cell & Molecular Biology and Toxicology; students in these majors 

are able to participate in this experimental course as an elective. The PI’s proposed plan of research will fully 

support the objectives for this internship. Many students will have completed the Biochemistry course (CHE 

315) concurrently with their lab research. CHE 315 course is coordinated and taught by the PI, preparing the 

students to participate meaningfully in the PI’s research. The biochemistry lab course is a series of skill-building 

experiments, culminating in a high-level final experiment; competency in performance, knowledge, and written 

communication skills must be demonstrated by students at this point. This is a solid preliminary training for 

laboratory research. All of the students are participants of the PRISM (Program for Research Initiatives for 

Science and Math) Program, established at John Jay College and endorsed by President Obama. Launched in 

2006, PRISM aims to provide science, technology, engineering, and math (STEM) students with faculty-led 

research experiences throughout their undergraduate career. It was created to improve student retention and 

graduation rates, increase student exposure and access to careers in STEM disciplines, and improve their 

competitiveness when applying to graduate schools. As a Hispanic-serving institution, John Jay is contributing to 

increased diversity in STEM fields. Students attend seminars and training programs to better prepare them for 

research. At the beginning of their junior year, students are matched with a faculty mentor. In addition to direct 

research experience, students are eligible for stipends for their research, conference travel grants, GRE 

preparatory help, advisement on graduate and professional school applications, and career planning. They can 

also look forward to guest lectures and informal seminars with research faculty, scientists, and other 

professionals. PRISM research experiences go beyond the traditional training received in the classroom; they 

include instruction on literature searches, project design, implementation, experimental sampling, data analysis, 

and scientific writing/presentation, as well as providing a community in which students are able to flourish. 

These experiences help to demonstrate that science is not exact, but an iterative process of questioning the world 

around us, providing students with the skills necessary to succeed in science beyond the classroom, and prepare 

them to join the global research community. All techniques and data analyses described in this proposal are an 

extension of laboratory classroom skills; the experiments lend themselves to a high degree of manageable 

compartmentalization and subsequent integration into the whole project. The research relies heavily on peers as 

co-researchers in its approach: experienced senior students are paired with incoming students. Since the 

experiments require a great deal of teaching/learning, and honing of technical skills, the PI is always accessible, 

working hands-on with individuals and the entire group. With this approach, students are immersed in science 

and incrementally learn techniques, quickly carrying high levels of responsibility.

Contact PD/PI: Domashevskiy, Artem

Research Strategy Page 43
Bibliography and References Cited

[1] Romano, J.A. Jr., Lukey, B.J., and Salem, H. (2007) Chemical Warfare Agents: Chemistry, 

Pharmacology, Toxicology, and Therapeutics, Second Ed., CRC Press. 

[2] Balali-Mood, M., Moshiri, M., and Etemad, L. (2013) Medical aspects of bio-terrorism. Toxicon 69, 131-

142. 

[3] Olsnes, S. (2004) The history of ricin, abrin and related toxins. Toxicon 44, 361-370. 

[4] Centers for Disease Control and Prevention (CDC). In Emergency Preparedness and Response: Ricin.

(http://emergency.cdc.gov/agent/ricin/).

[5] Barbieri, L., Battelli, M.G., and Stirpe, F. (1993) Ribosome-inactivating proteins from plants. Biochim. 

Biophys. Acta. 1154, 237-282. 

[6] Wang, P. and Tumer, N.E. (2000) Virus resistance mediated by ribosome inactivating proteins. Adv. 

Virus Res. 55, 325-355. 

[7] Walsh, M.J., Dodd, J.E., and Hautbergue, G.M. (2013) Ribosome-inactivating proteins: potent poisons 

and molecular tools. Virulence 4, 774-784. 

[8] Jimenez, A. and Vazquez, D. (1985) Plant and fungal protein and glycoprotein toxins inhibiting 

eukaryote protein synthesis. Annu. Rev. Microbiol. 39, 649-672. 

[9] Stirpe, F. (2004) Ribosome-inactivating proteins. Toxicon 44, 371-383. 

[10] Lam, S.K. and Ng, T.B. (2001) Hypsin, a novel thermostable ribosome-inactivating protein with 

antifungal and antiproliferative activities from fruiting bodies of the edible mushroom Hypsizigus 

marmoreus. Biochem. Biophys. Res. Commun. 285, 1071-1075. 

[11] Liu, R.S., Yang, J.H., and Liu, W.Y. (2002) Isolation and enzymatic characterization of lamjapin, the 

first ribosome-inactivating protein from cryptogamic algal plant (Laminaria japonica A). Eur. J. Biochem. 

269, 4746-4752. 

[12] Endo, Y. (1988) Mechanism of action of ricin and related toxins on the inactivation of eukaryotic 

ribosomes. In Immunotoxins (Frankel, A.E., Ed.), Kluwer Academic, Boston. pp 75-89. 

[13] Nielsen, K. and Boston, R.S. (2001) Ribosome-Inactivating Proteins: A Plant Perspective. Annu. Rev. 

Plant Physiol. Plant Mol. Biol. 52, 785-816. 

[14] Ferreras, J.M., Barbieri, L., Girbes, T., Battelli, M.G., Rojo, M.A., Arias, F.J., Rocher, M.A., Soriano, F., 

Mendez, E., and Stirpe, F. (1993) Distribution and properties of major ribosome-inactivating proteins 

(28 S rRNA N-glycosidases) of the plant Saponaria officinalis L. (Caryophyllaceae). Biochim. Biophys. 

Acta 1216, 31-42. 

[15] Domashevskiy, A.V. and Goss, D.J. (2015) Pokeweed Antiviral Protein, a Ribosome Inactivating 

Protein: Activity, Inhibition and Prospects. Toxins (Basel) 7, 274-298. 

[16] Stirpe, F. and Battelli, M.G. (2006) Ribosome-inactivating proteins: progress and problems. Cell Mol. 

Life Sci. 63, 1850-1866. 

[17] Barbieri, L., Polito, L., Bolognesi, A., Ciani, M., Pelosi, E., Farini, V., Jha, A.K., Sharma, N., Vivanco, 

J.M., Chambery, A., Parente, A., and Stirpe, F. (2006) Ribosome-inactivating proteins in edible plants 

and purification and characterization of a new ribosome-inactivating protein from Cucurbita moschata. 

Biochim. Biophys. Acta 1760, 783-792. 

[18] Johannes, L. and Romer, W. (2010) Shiga toxins--from cell biology to biomedical applications. Nature 

Rev. Microbiol. 8, 105-116. 

[19] Puri, M., Kaur, I., Perugini, M.A., and Gupta, R.C. (2012) Ribosome-inactivating proteins: current 

status and biomedical applications. Drug Discov. Today 17, 774-783. 

[20] Bergan, J., Dyve Lingelem, A.B., Simm, R., Skotland, T., and Sandvig, K. (2012) Shiga toxins. Toxicon

60, 1085-1107. 

[21] Stirpe, F. (2013) Ribosome-inactivating proteins: from toxins to useful proteins. Toxicon 67, 12-16. 

[22] Olsnes, S. and Pihl, A. (1973) Isolation and properties of abrin: a toxic protein inhibiting protein 

synthesis. Evidence for different biological functions of its two constituent-peptide chains. Eur. J. 

Biochem. 35, 179-185. 

[23] Olsnes, S. and Pihl, A. (1981) Chimeric toxins. Pharmacol. Ther. 15, 355-381. 

Contact PD/PI: Domashevskiy, Artem

References Cited Page 44
[24] Sandvig, K., Olsnes, S., and Pihl, A. (1976) Kinetics of binding of the toxic lectins abrin and ricin to 

surface receptors of human cells. J. Biol. Chem. 251, 3977-3984. 

[25] Olsnes, S. and Sandvig, K. (1988) How protein toxins enter and kill cells. Cancer Treat. Res. 37, 39-73. 

[26] Steeves, R.M., Denton, M.E., Barnard, F.C., Henry, A., and Lambert, J.M. (1999) Identification of three 

oligosaccharide binding sites in ricin. Biochemistry 38, 11677-11685. 

[27] van Deurs, B., Tonnessen, T.I., Petersen, O.W., Sandvig, K., and Olsnes, S. (1986) Routing of 

internalized ricin and ricin conjugates to the Golgi complex. J. Cell Biol. 102, 37-47. 

[28] Beaumelle, B., Alami, M., and Hopkins, C.R. (1993) ATP-dependent translocation of ricin across the 

membrane of purified endosomes. J. Biol. Chem. 268, 23661-23669. 

[29] Sandvig, K. and van Deurs, B. (1994) Endocytosis and intracellular sorting of ricin and Shiga toxin. 

FEBS Lett. 346, 99-102. 

[30] Knight, B. (1979) Ricin - a potent homicidal poison. Br. Med. J. 1, 350-351. 

[31] Bozza, W.P., Tolleson, W.H., Rosado, L.A., and Zhang, B. (2015) Ricin detection: tracking active toxin. 

Biotechnol. Adv. 33, 117-123. 

[32] Pita, R. and Romero, A. (2014) Toxins as Weapons: A Historical Review. Forensic Sci. Rev. 26, 85-96. 

[33] U.S. Department of Justice. Federal Bureau of Investigations (FBI). (2007) Terrorism 2002-2005. U.S. 

Department of Justice. p. 48. (https://www.fbi.gov/stats-services/publications/terrorism-2002-

2005/terror02_05.pdf).

[34] Craig, H.L., Alderks, O.H., Corwin, A.H., Dieke, S.H., and Karel, C.L. (1952) Preparation of toxic ricin. 

US Patent Office. Number 3,060,165.

[35] Melton-Celsa, A.R. (2014) Shiga Toxin (Stx) Classification, Structure, and Function. Microbiol. Spectr. 9, 

1-13. 

[36] O'Brien, A.D., Lively, T.A., Chen, M.E., Rothman, S.W., and Formal, S.B. (1983) Escherichia coli 

O157:H7 strains associated with haemorrhagic colitis in the United States produce a Shigella 

dysenteriae 1 (SHIGA) like cytotoxin. Lancet 1, 702. 

[37] Centers for Disease Control and Prevention (CDC). (2014) Multistate Outbreak of Shiga toxinproducing Escherichia coli O121 Infections Linked to Raw Clover Sprouts (Final Update). In E. coli 

Outbreaks. (http://www.cdc.gov/ecoli/2014/O121-05-14/index.html).

[38] Centers for Disease Control and Prevention (CDC). (2013) Multistate Outbreak of Shiga toxinproducing Escherichia coli O121 Infections Linked to Farm Rich Brand Frozen Food Products (Final 

Update). In E. coli Outbreaks. (http://www.cdc.gov/ecoli/2013/O121-03-13/index.html).

[39] Centers for Disease Control and Prevention (CDC). (2014) Multistate Outbreak of Shiga toxinproducing Escherichia coli O157:H7 Infections Linked to Ground Beef (Final Update). In E. coli 

Outbreaks. (http://www.cdc.gov/ecoli/2014/O157H7-05-14/index.html).

[40] Scallan, E., Hoekstra, R., Angulo, F.J., Tauxe, R.V., Widdowson, M.A., Roy, S.L., Jones, J.L., and 

Griffin, P.M. (2011) Foodborne illness acquired in the United States--major pathogens. Emerg. Infect. 

Dis. 17, 7-15. 

[41] Griffin, P.M. and Tauxe, R.V. (1991) The epidemiology of infections caused by Escherichia coli 

O157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome. Epidemiol. 

Rev. 13, 60-98. 

[42] Armstrong, G.L., Hollingsworth, J., and Morris, J.G. Jr. (1996) Emerging foodborne pathogens: 

Escherichia coli O157:H7 as a model of entry of a new pathogen into the food supply of the developed 

world. Epidemiol. Rev. 18, 29-51. 

[43] Abdul-Raouf, U.M., Beuchat, L.R., and Ammar, M.S. (1993) Survival and growth of Escherichia coli 

O157:H7 in ground, roasted beef as affected by pH, acidulants, and temperature. Appl. Environ. 

Microbiol. 59, 2364-2368. 

[44] Rangel, J.M., Sparling, P.H., Crowe, C., Griffin, P.M., and Swerdlow, D.L. (2005) Epidemiology of 

Escherichia coli O157:H7 outbreaks, United States, 1982-2002. Emerg. Infect. Dis. 11, 603-609. 

[45] Abdul-Raouf, U.M., Beuchat, L.R., and Ammar, M.S. (1993) Survival and growth of Escherichia coli 

O157:H7 on salad vegetables. Appl. Environ. Microbiol. 59, 1999-2006. 

Contact PD/PI: Domashevskiy, Artem

References Cited Page 45
[46] Russo, L.M., Melton-Celsa, A.R., Smith, M.J., and O'Brien, A.D. (2014) Comparisons of Native Shiga 

Toxins (Stxs) Type 1 and 2 with Chimeric Toxins Indicate that the Source of the Binding Subunit 

Dictates Degree of Toxicity. PLoS One 9, e93463. 

[47] Montanaro, L., Sperti, S., and Stirpe, F. (1973) Inhibition by ricin of protein synthesis in vitro. 

Ribosomes as the target of the toxin. Biochem. J. 136, 677-683. 

[48] Endo, Y., Mitsui, K., Motizuki, M., and Tsurugi, K. (1987) The mechanism of action of ricin and 

related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 

S ribosomal RNA caused by the toxins. J. Biol. Chem. 262, 5908-5912. 

[49] Girbes, T., Ferreras, J.M., Arias, F.J., and Stirpe, F. (2004) Description, distribution, activity and 

phylogenetic relationship of ribosome-inactivating proteins in plants, fungi and bacteria. Mini Rev. Med.

Chem. 4, 461-476. 

[50] Vater, C.A., Bartle, L.M., Leszyk, J.D., Lambert, J.M., and Goldmacher, V.S. (1995) Ricin A chain can 

be chemically cross-linked to the mammalian ribosomal proteins L9 and L10e. J. Biol. Chem. 270, 12933-

12940. 

[51] Hudak, K.A., Dinman, J.D., and Tumer, N.E. (1999) Pokeweed antiviral protein accesses ribosomes by 

binding to L3. J. Biol. Chem. 274, 3859-3864. 

[52] Monzingo, A.F. and Robertus, J.D. (1992) X-ray analysis of substrate analogs in the ricin A-chain active 

site. J. Biol. Chem. 227, 1136-1145. 

[53] Monzingo, A.F., Collins, E.J., Ernst, S.R., Irvin, J.D., and Robertus, J.D. (1993) The 2.5 Å structure of 

pokeweed antiviral protein. J. Mol. Biol. 233, 705-715. 

[54] Yan, X., Hollis, T., Svinth, M., Day, P., Monzingo, A.F., Milne, G.W., and Robertus, J.D. (1997) 

Structure-based identification of a ricin inhibitor. J. Mol. Biol. 266, 1043-1049. 

[55] Kurinov, I.V., Myers, D.E., Irvin, J.D., and Uckun, F.M. (1999) X-ray crystallographic analysis of the 

structural basis for the interactions of pokeweed antiviral protein with its active site inhibitor and 

ribosomal RNA substrate analogs. Protein Sci. 8, 1765-1772. 

[56] Jasheway, K., Pruet, J., Anslyn, E.V., and Robertus, J.D. (2011) Structure-based design of ricin 

inhibitors. Toxins (Basel) 3, 1233-1248. 

[57] Stein, P.E., Boodhoo, A., Tyrrell, G.J. Brunton, J.L., and Read, R.J. (1992) Crystal structure of the cellbinding B oligomer of verotoxin-1 from E. coli. Nature 355, 748-750. 

[58] Fraser, M.E., Chernaia, M.M., Lozlov, Y.V., and James, M.N. (1994) Crystal structure of the holotoxin 

from Shigella dysenteriae at 2.5 A resolution. Nat. Struct. Biol. 1, 59-64. 

[59] Domashevskiy, A.V., Miyoshi, H., and Goss, D.J. (2012) Inhibition of pokeweed antiviral protein 

(PAP) by turnip mosaic virus genome-linked protein (VPg). J. Biol. Chem. 287, 29729-29738. 

[60] Walsh, J.A. and Jenner, C.E. (2002) Turnip mosaic virus and the quest for durable resistance. Mol. Plant 

Pathol. 3, 289-300. 

[61] Suehiro, N., Natsuaki, T., Watanabe, T., and Okuda, S. (2004) An important determinant of the ability 

of Turnip mosaic virus to infect Brassica spp. and/or Raphanus sativus is in its P3 protein. J. Gen. Virol.

85, 2087-2098. 

[62] Fellers, J., Wan, J., Hong, Y., Collins, G.B., and Hunt, A.G. (1998) In vitro interactions between a 

potyvirus-encoded, genome-linked protein and RNA-dependent RNA polymerase. J. Gen. Virol. 79, 

2043-2049. 

[63] Li, X.H., Valdez, P., Olvera, R.E., and Carrington, J.C. (1997) Functions of the tobacco etch virus RNA 

polymerase (NIb): subcellular transport and protein-protein interaction with VPg/proteinase (NIa). J. 

Virol. 71, 1598-1607. 

[64] Hong, Y., Levay, K., Murphy, J.F., Klein, P.G., Shaw, J.G., and Hunt, A.G. (1995) A potyvirus 

polymerase interacts with the viral coat protein and VPg in yeast cells. Virology 214, 159-166. 

[65] Borgstrom, B. and Johansen, I.E. (2001) Mutations in pea seedborne mosaic virus genome-linked 

protein VPg after pathotype-specific virulence in Pisum sativum. Mol. Plant Microbe Interact. 14, 707-714. 

[66] Johansen, I.E., Lund, O.S., Hjulsager, C.K., and Laursen, J. (2001) Recessive resistance in Pisum 

sativum and potyvirus pathotype resolved in a gene-for-cistron correspondence between host and virus. 

J. Virol. 75, 6609-6614. 

Contact PD/PI: Domashevskiy, Artem

References Cited Page 46
[67] Keller, K.E., Johansen, I.E., Martin, R.R., and Hampton, R.O. (1998) Potyvirus genome-linked protein 

(VPg) determines pea seed-borne mosaic virus pathotype-specific virulence in Pisum sativum. Mol. Plant 

Microbe Interact. 11, 124-130. 

[68] Schaad, M.C., Lellis, A.D., and Carrington, J.C. (1997) VPg of tobacco etch potyvirus is a host 

genotype-specific determinant for long-distance movement. J. Virol. 71, 8624-8631. 

[69] Masuta, C., Nishimura, M., Morishita, H., and Hataya, T. (1999) A single amino Acid change in viral 

genome-associated protein of potato virus y correlates with resistance breaking in 'virgin a mutant' 

tobacco. Phytopathology 89, 118-123. 

[70] Weston, S.A., Tucker, A.D., Thatcher, D.R., Derbyshire, D.J., and Pauptit, R.A. (1994) X-ray structure 

of recombinant ricin A-chain at 1.8 A resolution. J. Mol. Biol. 244, 410-422. 

[71] Montfort, W., Villafranca, J.E., Monzingo, A.F., Ernst, S.R., Katzin, B., Rutenber, E., Xuong, N.H., 

Hamlin, R., and Robertus, J.D. (1987) The three-dimensional structure of ricin at 2.8 Å. J. Biol. Chem.

262, 5398-5403. 

[72] Yan, Y. and Marriott, G. (2003) Analysis of protein interactions using fluorescence technologies. Curr. 

Opin. Chem. Biol. 7, 635-640.

[73] Pierce, M.M., Raman, C.S., and Nall, B.T. (1999) Isothermal titration calorimetry of protein-protein 

interactions. Methods 19, 213-221.

[74] Bujalowski, W.M. and Jezewska, M.J. (2012) Fluorescence intensity, anisotropy, and transient dynamic 

quenching stopped-flow kinetics. Methods Mol. Biol. 875, 105-133.

[75] Khan, M.A. and Goss, D.J. (2012) Poly(A)-binding protein increases the binding affinity and kinetic 

rates on interaction of viral protein linked to genome with translation initiation factors eIFiso4F and 

eIFiso4F-4B complex. Biochemistry 51, 1388-1395.

[76] Ho, M.C., Sturm, M.B., Almo, S.C., and Schramm, V.L. (2009) Transition state analogues in structures 

of ricin and saporin ribosome-inactivating proteins. Proc. Nalt. Acad. Sci. USA 106, 20276-20281.

[77] Nishida, N. and Shimada, I. (2012) An NMR method to study protein-protein interactions. Methods Mol. 

Biol. 757, 129-137.

[78] Marintchev, A., Frueh, D., and Wagner, G. (2007) NMR methods for studying protein-protein 

interactions involved in translation initiation. Methods Enzymol. 430, 283-331.

[79] Chung, D.A. Zuiderweg, E.R., Fowler, C.B. Soyer, O.S., Mosberg, H.I., and Neubig, R.R. (2002) NMR 

structure of the second intracellular loop of the alpha 2A adrenergic receptor: evidence for a novel 

cytoplasmic helix. Biochemistry 41, 3596-3604.

[80] Zuiderberg, E.R. (2002) Mapping protein-protein interactions in solution NMR spectroscopy. 

Biochemistry 41, 1-7.

[81] O'Hare, M., Boberts, L.M., Thorpe, P.E., Waston, G.J., Prior, B., and Lord, J.M. (1987) Expression of 

ricin A chain in Escherichia coli. FEBS Lett. 216, 73-78.

[82] Piatak, M., Lane, J.A., Laird, W., Bjorn, M.J., Wang, A., and Williams, M. (1988) Expression of soluble 

and fully functional ricin A chain in Escherichia coli is temperature-sensitive. J. Biol. Chem. 263, 4837-4843.

[83] Becker, B. and Schmitt, M.J. (2011) Adapting Yeast as Model to Study Ricin Toxin A Uptake and 

Trafficking. Toxins (Basel) 3, 834-847.

[84] May, K.L., Yan, Q., and Tumer, N.E. (2013) Targeting ricin to the ribosome. Toxicon 69, 143-151.

[85] Uhlig, T., Kyprianou, T., Martinelli, F.G., Oppici, C.A., Heiligers, D., Hills, D., Calvo, X.R., and 

Verhaert, P. (2014) The emergence of peptides in the pharmaceutical business: From exploration to 

exploitation. EuPA Open Proteom. 4, 58-69.

[86] Fosgerau, K. and Hoffmann, T. (2015) Peptide therapeutics: current status and future directions. Drug 

Discov. Today 20, 122-128.

[87] Kaspar, A.A. and Reichert, J.M. (2013) Future directions for peptide therapeutics development. Drug 

Discov. Today 18, 807-817.

[88] Barbier, J., Bouclier, C., Johannes, L., and Gillet, D. (2012) Inhibitors of the cellular trafficking of ricin. 

Toxins (Basel) 4, 15-27. 

Contact PD/PI: Domashevskiy, Artem

References Cited Page 47
[89] Roday, S., Amukele, T., Evans, G.B., Tyler, P.C., Furneaux, R.H., and Schramm, V.L. (2004) Inhibition 

of ricin A-chain with pyrrolidine mimics of the oxacarbenium ion transition state. Biochemistry 43, 4923-

4933. 

[90] Legler, P.M., Brey, R.N, Smallshaw, J.E., Vitetta, E.S., and Millard, C.B. (2011) Structure of RiVax: a 

recombinant ricin vaccine. Acta Crystallogr. D. Biol. Crystallogr. 67, 826-830. 

[91] Smallshaw, J.E., Richardson, J.A., and Vitetta, E.S. (2007) RiVax, a recombinant ricin subunit vaccine, 

protects mice against ricin delivered by gavage or aerosol. Vaccine 25, 7459-7469. 

[92] Smallshaw, J.E., Firan, A., Fulmer, J.R., Ruback, S.L., Ghetie, V., and Vitetta, E.S. (2002) A novel 

recombinant vaccine which protects mice against ricin intoxication. Vaccine 20, 3422-3427. 

[93] Mayor, S. (2003) UK doctors warned after ricin poison found in police raid. BMJ 326, 126. 

[94] Occupational Safety and Health Administration (OSHA) (2003) Model Plans and Programs for the 

OSHA Bloodborne Pathogens and Hazard Communications Standards. (https://www.osha.gov/ 

Publications/osha3186.pdf).

[95] Lakowicz, J.R. (2007) Principles of Fluorescence Spectroscopy. Third Ed., Springer Science. 

[96] Baldwin, A.E., Khan, M.A., Tumer, N.E., Goss, D.J., and Friedland, D.E. (2009) Characterization of 

pokeweed antiviral protein binding to mRNA cap analogs: competition with nucleotides and 

enhancement by translation initiation factor iso4G. Biochim. Biophys. Acta 1789, 109-116. 

[97] Khan, M.A., Yumak, H., and Goss, D.J. (2009) Kinetic mechanism for the binding of eIF4F and 

tobacco Etch virus internal ribosome entry site rna: effects of eIF4B and poly(A)-binding protein. The J. 

Biol. Chem. 284, 35461-35470. 

[98] Cavanagh, J., Fairbrother, W.J., Palmer III, A.J., Rance, M., and Skelton, N. (2007) Protein NMR 

spectroscopy: Principles and practice. Second Ed., Academic Press, San Diego. 

[99] Goddard, T.D. and Kneller, D.G. (2008) Sparky - NMR Assignment and Integration Software. 

University of California, San Francisco, CA. (http://www.cgl.ucsf.edu/home/sparky/).

[100] Velazquez-Campoy, A., Leavitt, S.A., and Freire, E. (2004) Characterization of protein-protein 

interactions by isothermal titration calorimetry. Methods Mol. Biol. 261, 35-54. 

[101] Jelesarov, I. and Bosshard, H.R. (1999) Isothermal titration calorimetry and differential scanning 

calorimetry as complementary tools to investigate the energetics of biomolecular recognition. J. Mol. 

Recognit. 12, 3-18. 

[102] Khan, M.A., Miyoshi, H., Ray, S., Natsuaki, T., Suehiro, N., and Goss, D.J. (2006) Interaction of 

genome-linked protein (VPg) of turnip mosaic virus with wheat germ translation initiation factors 

eIFiso4E and eIFiso4F. J. Biol. Chem. 281, 28002-28010. 

[103] Khan, M.A., Miyoshi, H., Gallie, D.R., and Goss, D.J. (2008) Potyvirus genome-linked protein, VPg, 

directly affects wheat germ in vitro translation: interactions with translation initiation factors eIF4F and 

eIFiso4F. J. Biol. Chem. 283, 1340-1349. 

[104] Endo, Y. and Tsurugi, K. (1987) RNA N-glycosidase activity of ricin A-chain. Mechanism of action of 

the toxic lectin ricin on eukaryotic ribosomes. J. Biol. Chem. 262, 8128-8130. 

[105] Peattie, D.A. (1979) Direct chemical method for sequencing RNA. Proc. Natl. Acad. Sci. USA 76, 1760-

1764. 

[106] Tumer, N.E., Hwang, D.J., and Bonness, M. (1997) C-terminal deletion mutant of pokeweed antiviral 

protein inhibits viral infection but does not depurinate host ribosomes. Proc. Natl. Acad. Sci. USA 94, 

3866-3871. 

[107] Sturm, M.B. and Schramm, V.L. (2009) Detecting ricin: sensitive luminescent assay for ricin A-chain 

ribosome depurination kinetics. Anal. Chem. 81, 2847-2853. 

[108] Gentry, M.K. and Dalrymple, J.M. (1980) Quantitative microtiter cytotoxicity assay for Shigella toxin. J. 

Clin. Microbiol. 12, 361-366. 

[109] Schmitt, C.K., McKee, M.L., and O'Brien, A.D. (1991) Two copies of Shiga-like toxin II-related genes 

common in enterohemorragic Escherichia coli strains are responsible for the antigenic heterogeneity of 

the O157:H- strain. Infect. Immun. 59, 1065-1073. 

Contact PD/PI: Domashevskiy, Artem

References Cited Page 48
[110] Yee, A.A., Savchenko, A., Ignachenko, A., Lukin, J., Xu, X., Skarina, T., Evdokimova, E., Liu, C.S., 

Semesi, A., Guido, V., Edwards, A.M., and Arrowsmith, C.H. (2005) NMR and X-ray crystallography, 

complementary tools in structural proteomics of small proteins. J. Am. Chem. Soc. 127, 16512-16517. 

[111] Otwinowski, Z. and Minor, W. (1997) Processing of x-ray diffraction data collected in oscillation mode. 

Methods Enzymol. 276, 307-326. 

[112] Marley, J., Lu, M., and Bracken, C. (2001) A method for efficient isotopic labeling of recombinant 

proteins. J. Biomol. NMR 20, 71-75. 

[113] Abagyan, R. and Argos, P. (1992) Optimal protocol and trajectory visualization for conformational 

searches of peptides and proteins. J. Mol. Biol. 225, 519-532. 

[114] Smellie, A., Teig, S.L., and Towbin, P. (2004) Poling: Promoting conformational variation. J. Comp. 

Chem. 16, 171-187. 

[115] Gohlke, H. and Klebe, G. (2002) Approaches to the description and prediction of the binding affinity 

of small-molecule ligands to macromolecular receptors. Angew. Chem. Int. Ed. Engl. 41, 2644-2676. 

[116] Kroemer, R.T. (2007) Structure-based drug design: docking and scoring. Curr. Protein Pept. Sci. 8, 312-

328. 

[117] Rarey, M., Kramer, B., Lengauer, T., and Klebe, G. (1996) A fast flexible docking method using an 

incremental construction algorithm. J. Mol. Biol. 261, 470-489. 

[118] Jones, G., Willet, P., Glen, R.C., Leach, A.R., and Taylor, R. (1997) Development and validation of a 

genetic algorithm for flexible docking. J. Mol. Biol. 267, 727-748. 

[119] Peitsch, M.C., Schwede, T., Diemand, A., and Guex, N. (2002) Protein Structure Prediction by 

Comparison: Homology-Based Modeling. In Current Topics in Computational Molecular Biology. Jiang, T. Xu, 

Y., and Zhang, M.Q. Eds., MIT Press, Cambridge, MA. pp 449-466. 

[120] Zimmer, R. and Lengauer, T. (2002) Bioinformatics - from Genomes to Drugs. Lengauer, T. Ed., WileyVCH, New York. pp 237-313. 

[121] Welch, W., Ruppert, J., and Jain, A.N. (1996) Hammerhead: fast, fully automated docking of flexible 

ligands to protein binding sites. Chem. Biol. 3, 449-462. 

[122] Kuntz, I.D., Blaney, J.M, Oatley, S.J., Langridge, R., and Ferrin, T.E. (1982) A geometric approach to 

macromolecule-ligand interactions. J. Mol. Biol. 161, 269-288. 

[123] Allen, W.J., Balius, T.E., Mukherjee, S., Brozell, S.R., Moustakas, D.T., Lang, P.T., Case, D.A., Kuntz, 

I.D., and Rizzo, R.C. (2015) DOCK 6: Impact of new features and current docking performance. J. 

Comput. Chem. 36, 1132-1156. 

[124] Avanti, C. (2012) Innovative Strategies for Stabilization of Therapeutic Peptides in Aqueous 

Formulations. Dutch Top Inst. Parma (https://www.tipharma.com/fileadmin/user_upload/Theses/ 

PDF/Christina_Avanti_D6-202.pdf).

[125] Zheng, L., Baumann, U., and Reymond, J.L. (2004) An effective one-step site-directed and sitesaturation mutagenesis protocol. Nucleic Acids Res. 32, e115. 

[126] Kunkel, T.A. (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. 

Natl. Acad. Sci. USA 82, 488-492. 

[127] Sugimoto, M., Esaki, N., Tanaka, H., and Soda, K. (1989) A simple and efficient method for the 

oligonucleotide-directed mutagenesis using plasmid DNA template and phosphorothioate-modified 

nucleotide. Anal. Biochem. 179, 309-311. 

[128] Taylor, J.W., Ott, J., and Eckstein, F. (1985) The rapid generation of oligonucleotide-directed mutations 

at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 13, 8765-8785. 

[129] Vandeyar, M.A., Weiner, M.P., Hutton, C.J., and Batt, C.A. (1988) A simple and rapid method for the 

selection of oligodeoxynucleotide-directed mutants. Gene 65, 129-133. 

Contact PD/PI: Domashevskiy, Artem

References Cited Page 49
		

	 	

Anthony Carpi, PhD 

Associate Provost & Dean of Research 

Office for the Advancement of Research 

New York, NY 10019

 212.621.3735 

acarpi@jjay.cuny.edu 

June 7, 2016 

Proposal Review Committee 

Academic Research Enhancement Award 

National Institutes of Allergy and Infectious Diseases 

National Institute of Health 

Dear Review Committee, 

I am writing this letter of recommendation in support of Dr. Artem Domashevskiy and his 

application to the National Institutes of Health Academic Research Enhancement Award. I am 

happy to support him in the project entitled “Development of a novel inhibitor of Ricin: A 

potential therapeutic lead against deadly Shiga and related toxins.” The proposal focuses on the 

inhibition of the poison ricin and a related Shiga toxin produced by an enteric pathogenic 

bacteria by a viral peptide. Dr. Domashevskiy directs a research laboratory in the Department of 

Sciences at John Jay College with undergraduate and graduate students assisting him in his 

investigations of plants defense mechanisms. His proposed is a collaborative effort of his 

students and professional colleagues, and is an important contribution to his on-going success 

at John Jay College. Dr. Domashevskiy’s laboratory is fully equipped with the facilities necessary 

for the successful completion of the project. I am confident that the results of this study will 

provide important new insights into the design of novel peptide inhibitors of plant phytotoxins. 

This research has excellent potential for success, and will enhance the environment of research 

and education at John Jay College. 

I enthusiastically support his application the Award. Please feel free to contact me should you 

have any additional questions. 

Sincerely, 

Anthony Carpi, PhD 

Contact PD/PI: Domashevskiy, Artem

Letters of Support Page 50
Contact PD/PI: Domashevskiy, Artem

Letters of Support Page 51
Contact PD/PI: Domashevskiy, Artem

30

June 8, 2016 

Proposal Review Committee 

Academic Research Enhancement Award 

National Institutes of Allergy and Infectious Diseases 

National Institute of Health 

Dear Review Committee, 

I am writing in support of the application submitted by Dr. Artem Domashevskiy who is applying for a 

National Institutes of Health Academic Research Enhancement Award. 

I am pleased to be able to collaborate with Artem on his project entitled “Development of a novel 

inhibitor of ricin: A potential therapeutic lead against deadly Shiga and related toxins.” The research 

deals with the studies of a viral peptide and its inhibition of toxic ribosome inactivating proteins. 

My laboratory has worked on ricin A-chain isolated from castor beans, expressed in E. coli and

saporin L3 protein from soapwort leaves, expressed in excretion yeast vectors. We have developed 

exquisite assay systems for these difficult enzymes. 

We are pleased to share these reagents with Dr. Domashevskiy for use in his research program. My 

laboratory is pleased to provide plasmids, reagents, and research advice to support Dr. 

Domashevskiy’s project. I am confident that the results of his study will provide interesting and 

important new insights into inhibition of ricin and Shiga toxin by a viral genome protein. 

I enthusiastically support Dr. Domashevskiy’s application for the Academic Research Enhancement 

Award and look forward to our collaboration. 

Please feel free to contact me should you have any additional questions. 

Sincerely, 

Vern L. Schramm 

Science at the heart of medicine

Vern L. Schramm

Professor & Ruth Merns Chair

Department of Biochemistry

Jack & Pearl Resnick Campus

1300 Morris Park Ave., Bronx, NY 10461 

718.430.2813 fax 718.430-8565

vern@aecom.yu.edu

Letters of Support Page 52
Contact PD/PI: Domashevskiy, Artem

Letters of Support Page 53
Contact PD/PI: Domashevskiy, Artem

DEPARTMENT of CHEMISTRY & BIOCHEMISTRY

The City College ofNew York

Center for Discovery and Innovation David Jeruzalmi, PhD

City College of New York Professor

Convent Avenue at 138th Street V: 212.650.6062

New York, NY 10031 F: 212.650.7948

E: dj@ccny.cuny.edu

June 22, 2016 

Proposal Review Committee 

Academic Research Enhancement Award 

National Institutes of Allergy and Infectious Diseases 

National Institute of Health 

Dear Review Committee, 

I am pleased to collaborate with Dr. Artem Domashevskiy on his project entitled 

“Development of a Novel Inhibitor of Ricin: A Potential Therapeutic Lead against Deadly 

Shiga and Related Toxins.” The proposal focuses on the inhibition of ribosome 

inactivating proteins, specifically ricin from castor bean plant and Shiga toxin from 

enteric bacteria by a viral peptide from turnip mosaic virus. 

My research group laboratory is interested in the molecular mechanisms that 

underlie the faithful transmission of genetic information. We are currently focused on 

two areas, the machinery associated with DNA replication and 2) nucleotide excision 

repair. The goal of research in my laboratory is to provide a structural view of these 

"machines" and, by concomitant application of biochemical approaches, to provide a 

fundamental understanding of the underlying mechanisms. Our approaches are 

primarily structural, X-ray crystallography or cryo-electron microscopy. 

My laboratory will aid Dr. Domashevskiy in the crystallization of the RTA-VPg 

complex, recording of X-ray diffraction, and data analysis. My research group has 

extensive experience in macromolecular structure determination using X-ray 

crystallography. The proposed crystallization trials will be greatly facilitated by 

availability of a complete automated crystallization facility at the CUNY-Advanced 

Science Research Center, just steps away from my research laboratory. The available 

instruments include: 2 Gryphon protein crystallization robots (at 25°C and 4°C), 2 

Minstrel crystallization tray imagers (at 25°C and 4°C), and an Alchemist crystallization 

screen-maker. Lastly, my research group is actively measuring X-ray ray diffraction and 

determining crystal structures. As such, when diffraction data for the targeted entities 

becomes available, structure determination will proceed smoothly. 

I am confident that the results of this study will provide important new insights 

into the design of novel peptide inhibitors of cytotoxic RIPs. 

Sincerely, 

David Jeruzalmi, Ph.D. 

Letters of Support Page 54
Contact PD/PI: Domashevskiy, Artem

Letters of Support Page 55
AUTHENTIFICATION OF KEY BIOLOGICAL AND CHEMICAL RESOURCES

All key resources for this proposal will be authenticated to enhance the reproducibility of our results, as 

appropriate and according to NIH policy. 

Key Biological Resources that will be utilized in this proposal include: 

Cell lines: DH5α (E. coli competent cells), BL21(DE3) (E. coli competent cells), Vero cells (from kidney 

epithelial cells extracted from African green monkey).

Cell lines MAX Efficiency® DH5αTM Competent Cells will be purchased from ThermoFisher Scientific

(InvitrogenTM distributer; catalog number 18258012), and come with the company’s authentication certificate.

Cell lines One Shot® BL21(DE3) Chemically Competent E. coli will be purchased from ThermoFisher 

Scientific (InvitrogenTM distributer; catalog number C600003), and come with the company’s authentication 

certificate.

Cell lines Vero Cell Line will be purchased from Sigma-Aldrich® (84113001-1VL) and come with the 

company’s authentication certificate.

Contact PD/PI: Domashevskiy, Artem

Authentication of Key Biological and/or Chemical Resources Page 56