bluuebunny/crossref_metadata_2025 huggingface dataset:
CRISPR‐mediated removal of antibiotic resistance genes in Enterococcus faecalis populations
Authors: Rodrigues Marinelle , Hullahalli Karthik , Palmer Kelli
Published: November 2023

Multidrug resistant infections caused by Enterococcus faecalis are serious threats in hospitals where immunocompromised individuals are at elevated risk of infection. A major cause for the rise of drug resistance in E. faecalis is the horizontal transfer of antibiotic resistance genes on mobile genetic elements including integrative and conjugative elements and the species‐specific pheromone‐responsive plasmids. The transfer of these potentially beneficial mobile elements among the population can be impeded by genome defense systems such as the prokaryotic adaptive immune system CRISPR‐Cas. However, multidrug‐resistant strains of E. faecalis circumvent this by compromising their genome defense systems, leaving them vulnerable to invasion by mobile DNA. In this study, the cas genes of a native E. faecalis CRISPR‐Cas system were introduced into a MDR strain, leading to the reactivation of host genome defenses. When self‐targeting CRISPR spacers were employed on conjugative elements in heterogenous populations of E. faecalis, we found that certain antibiotic resistance genes could be selectively removed from the population under pressure to maintain the conjugative element. Taken together, this study demonstrates that CRISPR‐mediated alteration of E. faecalis population structure may be a viable option for depleting undesirable traits from E. faecalis human gut populations.

Support or Funding Information

This project is supported by R01AI116610 to KP.
Conserved DNA motifs in the type II‐A CRISPR leader region
Authors: Rajan Rakhi , Van Orden Mason , Klein Peter , Kesavan Babu , Najar Fares
Published: November 2023

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR‐associated (Cas) proteins consist of RNA‐protein complexes that provide bacteria and archaea sequence‐specific immunity against bacteriophages and conjugative plasmids. The bacteria or archaea become immune to phage or plasmid infections by inserting short pieces of the intruder DNA site‐specifically into the leader‐repeat (repetitive units of CRISPR RNA gene) junction, in a process called adaptation. The hypothesis of the present work is that sequence/structural motifs present in the leader‐repeat junction recruits type‐specific Cas proteins for successful adaptation. To test this, we analyzed 167 type IIA CRISPR loci belonging to different bacterial genera. Our dataset consisted of 150 nucleotides of the CRISPR leader, the first repeat, and the protein sequences of Cas1, Cas2, Cas9, and Csn2 proteins. After performing multiple sequence alignments using MUSCLE, phylogenetic trees were generated in UGENE. Our results indicate two highly conserved DNA motifs at the 3′ leader end; 5′‐ATTTGAG‐3′ (indicated as group 1) and 5′‐CTRCGAG‐3′ (indicated as group 2). A third group with a very short 5′‐CG‐3′ DNA conservation at the 3′ leader end is observed mostly in Lactobacilli. Interestingly, the 3′‐leader end of group 1 is associated with the CRISPR‐1 locus of Streptococcus thermophilus DGCC7710, while that of group 2 is associated with the CRISPR‐3 locus of S. thermophilus DGCC7710. Since the CRISPR loci 1 and 3 of S. thermophilus DGCC7710 have been established as two distinct type IIA locus, our results demonstrate the association of these two loci with distinct 3′‐leader end sequences. A phylogenetic analysis of the repeat and Cas proteins revealed segregation reflecting the 3′ leader end conservation and pointed to the coevolution of the leader region and the CRISPR components. Previous studies have shown that the 3′‐leader end sequences are important for adaptation in S. thermophilus (group 1) and S. pyogenes (group 2) type IIA systems. Our results expanded this and demonstrated that the 3′‐leader end sequence conservation is more universally conserved covering several (~50 in this study) bacterial genera. Studies are ongoing to experimentally prove the importance of the 3′ leader end sequences and its co‐relation to locus‐specific Cas proteins. Similar studies can be used to identify conserved leader sequences in other CRISPR types and sub‐types.

Support or Funding Information

Research was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under the grant number P20GM103640.
How the Splicing Factors Cus2 and Prp5 Influence Global Splicing Efficiency in Yeast
Authors: Clark Arthur , Norden Ian , Hoskins Aaron A.
Published: November 2023

Splicing is among the most complex biochemical processes in eukaryotic gene expression. Splicing involves the removal of non‐coding introns from pre‐mRNA and the ligation of exons to form mRNA. Splicing is catalyzed by an aggregation of small nuclear ribonucleoproteins (snRNPs) called the spliceosome. Cus2 and Prp5 are two proteins involved in the early steps of splicing. Cus2 is an RNA binding protein that modules conformation of the U2 snRNA. Prp5 is an RNA‐dependent ATPase which is essential for yeast spliceosome assembly by facilitating U2 snRNA basepairing to the intron. ATP hydrolysis by Prp5 is necessary for removal of Cus2 from the U2 snRNP prior to this basepairing, which would imply an antagonistic relationship between Prp5 and Cus2. We are investigating this relationship using RNA‐Seq to study splicing of endogenous yeast introns in Prp5 and Cus2 mutant strains. The initial results indicate that deletion of Cus2 decreases the global splicing efficiency of yeast, particularly the splicing of non‐ribosomal protein pre‐mRNAs. To investigate this further, we have designed RNA‐seq experiments to further test the interplay of Cus2 and Prp5 by employing strains that either contain Cus2 or in which Cus2 was deleted and either contain a wild type Prp5 or an ATPase‐deficient Prp5 mutant. These experiments will identify which yeast pre‐mRNAs are most dependent and most independent of Cus2 and ATP hydrolysis by Prp5 for their splicing.
Structural Characterization of the Recognition of U6 snRNA by the Yeast U6 Biogenesis Protein Usb1
Authors: DeLaitsch Andrew T , Didychuk Allison L , Montemayor Eric J , Larson Matthew A , Lucarelli Stefani , Butcher Samuel E
Published: November 2023

The spliceosome is an essential molecular machine responsible for the removal of non‐coding introns from precursor messenger RNA in eukaryotic cells. An essential component of the catalytically active spliceosome, the U6 snRNA, must undergo a biogenesis pathway prior to incorporation into the spliceosome. Maturation of U6 snRNA is mediated in part by the highly conserved U six biogenesis protein (Usb1), a 3′‐5′ exoribonuclease responsible for the post‐transcriptional removal of uridines from U6 RNA. In addition, the enzyme is responsible for leaving behind a specific 3′ end modification in the form of a terminal phosphate, which is required for high affinity binding of a component of the U6 snRNP, the Lsm2–8 protein complex. Mutations in the gene encoding Usb1 give rise to the autosomal recessive disease poikiloderma with neutropenia (PN) in humans. I expressed and purified Usb1 from S. cerevisiae, which crystallized and diffracted X‐ray to 1.8 Å. The structure is remarkably similar to the structure of human Usb1 despite less than 20% sequence identity. A sulfate ion occupies the active site in a manner that may mimic coordination to the terminal phosphate of the RNA. Understanding how Usb1 recognizes RNA will give us insight into the mechanism of Usb1 processing and its role in U6 snRNA biogenesis.

To biochemically characterize Usb1, I incubated recombinant Usb1 with a fluorescent RNA and monitored exoribonuclease activity via denaturing polyacrylamide gel electrophoresis. The assays show that activity is inactivated upon mutation of active site residues. In addition, fluorescence polarization binding experiments were used to monitor binding of RNA with variants of Usb1. The results of these experiments will help determine conditions most suitable for structural determination by NMR, SAXS, or x‐ray crystallography methods. I will present our progress towards determining a co‐structure of Usb1 with RNA in its active site with the goal of characterizing how the enzyme recognizes and subsequently processes U6 RNA for incorporation into the spliceosome.
Exploring Dib1's Role in pre‐mRNA Splicing
Authors: Schreib Christian Cody , Hernandez Cody , Bowman Emily , Lucas Amber , Maeder Corina
Published: November 2023

Pre‐messenger RNA splicing is a molecular process conserved throughout all eukaryotic organisms in which non‐protein coding regions of the pre‐messenger RNA are removed. This process is facilitated by the spliceosome, a large multi‐component molecular machine made of five small nuclear RNAs and ~100 proteins. One such protein is Dib1, a small protein in the U4/U6‐U5 triple snRNP that has been shown to be essential to cell viability and splicing. Previous work suggests that Dib1 leaves the splicing machinery once it assembles, and recent cryo‐EM structures indicate that Dib1 is housed in a central location in the U4/U6‐U5 triple snRNP.

To better understand what role Dib1 plays in splicing, we aimed to identify essential regions of the protein by identifying temperature sensitive mutations. Conserved residues found in hydrophobic pockets, basic regions, and loop regions of Dib1 were mutated to alanines utilizing site‐directed mutagenesis. These Dib1 mutants were transformed into a S. cerevisiae dib1Δ strain. Growth phenotypes of these yeast were surveyed using a serial dilution growth assay. Two Dib1 mutations caused decreased cell viability at 37°C. One of these mutations is located in a hydrophobic pocket and the other in an external loop region. The temperature sensitive mutations were further characterized by assaying their effect on splicing efficiency through in vitro splicing assays. Both mutations were found to cause severe splicing defects, blocking splicing prior to the first step. We then assayed the assembly of the spliceosome in the presence of these temperature sensitive Dib1 mutants through native gel analysis. Because splicing is an intricate process that involves many complexes coming on and off the pre‐mRNA, knowing the specific complexes on the pre‐mRNA can tell us when in the process of spliceosome assembly the Dib1 mutations had an effect, and in turn tell us when in the spliceosome assembly Dib1 is essential. The results of these assays suggest that Dib1 plays a vital role in the binding of the U4/U6‐U5 triple snRNP to the pre‐mRNA.

Support or Funding Information

Arnold and Mabel Beckman Foundation, NIH NIGMS R15GM120720, Welch Foundation
Design of a Gene Transfer Vector to Deliver a Stabilized Anti‐EGFR RNA Aptamer to the Glioblastoma Microenvironment
Authors: Parikh Sachin , Hicks Martin J
Published: November 2023

Glioblastoma multiforme (GBM) is an incurable and aggressive type of brain tumor. It is the most common central nervous system (CNS) malignancy with a median survival of only 14 months. The epidermal growth factor receptor (EGFR) is a type of tyrosine kinase receptor (TKR) dysregulated in about 60% of GBM tumors. EGFR amplification and over‐expression leads to angiogenesis and uncontrolled growth and proliferation of GBM. Although a great deal is known about the biology exhibited by EGFR‐activated GBM, the application of therapies against the biologic processes is limited by the blood‐brain barrier, which restricts systemically administered therapies from reaching the brain. We have created an in vivo tissue culture model to develop a novel strategy to bypass these barriers by developing a gene transfer vector to deliver the genetic sequences of a verified anti‐EGFR RNA therapy aptamer that binds with high affinity to EGFR. To stabilize the structure, we have added inactivated hammerhead ribozymes, and to promote extracellular movement of the aptamer, we use an extracellular RNA “exRNA” localization element. Methodologies include RNA isolation, reverse‐transcription and PCR to detect changes in EGFR transcript expression, as well as ELISA to measure changes in the phosphorylated and active form of EGFR. With the revelation that GBM leads to an abundant increase in exosomes and microvesicles, this strategy may have implications for future therapies directed against GBM.
Investigation of ck/MyosinVIIA interactors using affinity tagged C‐terminal binding domains
Authors: Halverson Thomas M , Sallee Jennifer L
Published: November 2023

Actin‐based protrusions play a variety of roles in cells from motility via filopodia to increasing surface area in microvilli to providing the molecular basis of hearing in humans and other vertebrates with stereocilia. Loss of function mutations in the unconventional myosin motor protein Myosin VIIA result in shortened stereocilia which lack correct polarity of the protrusion. Theses mutations are seen in seventy‐five percent of patients afflicted with Usher Syndrome, the leading genetic cause of hereditary deaf‐blindness. Drosophila melanogaster contain a homologous protein to Myosin VIIA called crinkled (ck/MyoVIIA). Mutations in ck/MyoVIIA result in shortened, split, and malformed hairs and denticles, actin based protrusions similar to vertebrate microvilli and stereocilia. Our project aims to identify proteins that interact with ck/MyoVIIA to help determine the molecular mechanism of ck/MyoVIIA in actin‐based protrusion formation. In this study, we are investigating the binding of ck/MyoVIIA to Cad99C. Cad99C is the Drosophila homolog to Protocadherin 15, a protein necessary in the formation of stereocilia that is known to interact with MyoVIIA in humans. In addition, Cad99C has been shown to interact with ck/MyoVIIA in the formation of follicle epithelial cells in Drosophila oocytes. We want to investigate whether this interaction occurs in embryos due to the known effect of loss of ck/MyoVIIA on actin‐based denticle formation in embryos. To test this protein interaction we used a transgenic Drosophila expressing the C‐terminal tail binding domains (MyTH4, FERM, and SH3) of ck/MyoVIIA fused to an N‐terminal triple affinity tag (FLAG, His, STREPII tags). The triple tag ck‐tail and HA‐Cad99C were expressed using a Squash‐GAL4 driver and isolated using a Talon metal affinity pull‐down procedure for the HIS tag. Proteins were separated using SDS‐page gel electrophoresis and identified using anti‐FLAG antibody for the ck‐tail and anti‐HA antibody for Cad99C.

Support or Funding Information

Beta Beta Beta Undergraduate Research Grant
Nanobody Mediated Neutralization of Listeria monocytogenes
Authors: Brooks Cory , Huh Ian , Toride Moeko , Shenai Akhelish
Published: November 2023

Listeriosis represents a significant public health concern, as consumption of food contaminated with Listeria monocytogenes can result in fatal meningitis and termination of the fetus during pregnancy. A series of recent outbreaks originating from foods historically considered to be low risk for Listeria infection highlights the need for novel strategies to improve food safety. The first step in bacterial invasion of host cells is interaction of Listeria cell surface proteins called Internalins with host cell receptors triggering receptor‐mediated endocytosis. Internalin B (InlB) binds the receptor tyrosine kinase cMET, an interaction that has been demonstrated to be crucial for bacterial invasion of the placenta. Given the importance of this interaction for bacterial virulence it represents a target for therapeutic intervention. We have isolated several nanobodies, the single domain antibodies derived from the unique heavy‐chain antibodies of camels, which bind to InlB with high affinity. A combination of gentamicin protection assays and florescent microscopy demonstrate that InlB specific nanobodes can block the invasion of Listeria cell in vitro. Furthermore, a high resolution X‐ray structure of one of the nanobodies in complex with InlB reveals that the nanobody binds the cMet interaction site, acting as a competitive inhibitor preventing bacterial invasion. The results presented point to the potential of nanobodies as a novel class of therapeutics for the prevention of Listeriosis.

Support or Funding Information

Projected is funded by a NIH SCORE (SC3) grant (SC3GM112532)
Functional Role of the N‐terminal Region of bHLH TWIST Proteins in the DNA‐binding Activity of ADD1/SREBP1c
Authors: Rodriguez Yacidzohara , Cadilla Carmen L
Published: November 2023

TWIST1 and TWIST2 proteins are key bHLH transcription factors involved in the inhibition of differentiation of mesenchymal cell lineages during early stages of embryogenesis. Besides using the common bHLH domain for protein‐protein interactions with other proteins, they are also able to use their N‐ and C‐terminus domains. Although the transcriptional regulation of interacting proteins by use of their bHLH and C‐terminus has been extensively studied, there is little information that directly compares the N‐terminus of both Twist proteins interacting with a common partner. ADD1/SREBP1c is a transcription activator whose activity has been shown to be repressed by TWIST2, mainly by reducing its DNA binding to target sequences. To determine whether TWIST2 and TWIST1 may employ their N‐terminal regions to interact with ADD1, we used truncated TWIST2 (Q65X) and TWIST1 (Q107X) proteins containing only their N‐terminal regions. EMSA analysis showed reduced DNA binding of ADD1 homodimers when co‐expressed with Q65X, which suggests that TWIST2 uses its N‐terminus to interact with ADD1. In contrast, the DNA binding of ADD1 was not affected when co‐expressed with mutant TWIST1 Q107X, suggesting that the N‐terminus of TWIST1 does not interact with ADD1. To determine the region of TWIST2 responsible for the interaction with ADD1, we performed deletion mutagenesis of two conserved sub‐motifs found in the N‐terminus: SSSPVSP (Twist2ΔA) and SEEE (Twist2ΔB). EMSA analysis showed ADD1 DNA binding as homodimers when co‐expressed with Twist2ΔB but not when Twist2ΔA was co‐expressed, suggesting that TWIST2 uses the SEEE sub‐motif to interact with ADD1. To determine whether the sequence differences in the N‐terminal region of TWIST2 and TWIST1 account for the functional difference in protein‐protein interactions with ADD1, deletion mutagenesis of TWIST1's glycine‐rich regions was conducted. EMSA results showed that like WT TWIST2, removal of TWIST1's both glycine‐rich regions allows interaction between TWIST1 and ADD1, as seen by the loss of ADD1's binding to the DNA. To determine which of the two glycine‐rich regions is used for the interaction with ADD1, we deleted either the first glycine‐rich region (Twist1ΔGly1) or second glycine‐rich region (Twist1ΔGly2). EMSA results demonstrate that when ADD1 is co‐expressed with Twist1ΔGly1, the DNA binding activity of ADD1 is greatly increased. However, when co‐expressed with Twist1ΔGly2, DNA binding activity of ADD1 is reduced. This suggests that the first glycine‐rich region of TWIST1 could be responsible for the interaction with ADD1. In conclusion, TWIST1 and TWIST2 proteins use different regions of their Amino‐terminus to differentially regulate the DNA‐binding activity of ADD1/SREBP1c.

Support or Funding Information

Research was supported by grants: G12MD007600 (RCMI) and R25GM061838 (RISE)
Exploring a Possible Role in Copper Transport and Storage for the Vitamin Carrier, Riboflavin Binding Protein
Authors: Smith Sheila , Benore Marilee
Published: November 2023

Riboflavin Binding Protein (RBP) has been shown previously to bind copper in vitro in a one to one molar ratio. The binding site, characterized by EPR, EXAFS, ESEEM and ENDOR spectropcopies is a well ordered Type II copper site with one histidine ligand and 2–3 oxygenic ligands. Copper‐loaded RBP was dialyzed with the egg protein phosvitin, previously shown to bind multiple equivalents of copper. The proteins were separated by FPLC and the copper content was measured by graphite furnace AA spectroscopy. All of the copper was transferred to the phosvitin. Relative binding constants of the two proteins were determined. We present a hypothesis for the role of copper binding and protein‐protein interaction in copper transport into the avian egg.
Investigating the Role of a Dual Motif in DNA Binding by PAX3‐FOXO1
Authors: Weatherspoon Amber , Evans Ronise , Cherlin Tess , Bratton Melyssa , Johanson Kelly
Published: November 2023

Rhabdomyosarcoma (RMS) is a common soft tissue sarcoma that can be divided into two main categories. Embryonal Rhabdomyosarcoma (ERMS) usually affects children in the first five years of life and has not been linked to the presence of specific gene fusions. Alveolar Rhabdomyosarcoma (ARMS), which is more aggressive, typically affects all age groups equally but a larger portion of RMS occur in older children. The more aggressive form of ARMS is primarily characterized by a reciprocal (2;13) (q35; q14) translocation, which results in the fusion of the N‐terminus of PAX3 and the C‐terminus of FOXO1. PAX3‐FOXO1 mediated gene activation leads to ARMS development has been linked to several events involved in ARMS development, although all targets of this fusion protein have not been identified. A search of an updated ChIP‐seq screen of 3881 PAX3‐FOXO1 putative binding sites revealed that 36% contained both a PAX3 paired domain and FOXO1 recognition sequence within proximity. In vitro examination of a PAX3‐FOXO1 dual motif indicates that mutation of the FOXO1 site or residues in the FOXO1 DNA‐binding domain affect both binding and activation of these sequences. Binding sites for other transcription factors overlap or surround several of these dual motifs, indicating the need to examine potential transcriptional complexes formed by PAX3‐FOXO1. Understanding the binding specificities and activities of this fusion protein will allow for a better understanding of the transcription factor which may lead to future therapeutic remedies.

Support or Funding Information

NIH BUILD RL5GM118966‐02, NIH RCMI 2G12MD007595‐06, NIH BUILD TL4GM118968
Elucidating Met18 Functionality in Iron‐Sulfur Cluster Biogenesis
Authors: Lee Claudia Yejin , Vo Amanda , Cosman Jessica , Perlstein Deborah
Published: November 2023

The Cytosolic Iron‐Sulfur Cluster Assembly Pathway (CIA pathway) is a highly conserved and essential pathway that assembles and delivers iron sulfur clusters to recipient proteins in all eukaryotic organisms. Several FeS proteins require iron‐sulfur clusters for vital processes such as DNA metabolism, iron homeostasis, and cellular respiration. These apo‐FeS proteins (termed targets) are identified by a targeting complex composed of Met18, Cia2, and Cia1 in yeast. Met18 has a superhelical structure composed of HEAT (Huntington, Elongation Factor 3, PR65/A, TOR) domain repeats, which structurally in other protein is responsible for identifying several protein substrates. Within Met18's HEAT repeat domains, previous literature has shown two highly conserved regions at the N and C terminus. We hypothesize that these regions could play a role in targeting complex and/or target binding. To investigate Met18's functionality in vitro, we developed the first methods to effectively access large quantities of Met18. We then probed for interactions using affinity copurification and determined that Met18 and Cia2 directly interact and that a sub complex of Cia2‐Met18 can associate with model apo‐target, Rad3. To determine whether the N or C terminus was sufficient for Cia2 and/or Rad3 binding, N‐terminal and C‐terminal truncation mutants (Met18 NT, Met18 CT) were created. Through copurification experiments with Met18 NT and Cia2, we found the N‐terminus alone is insufficient to form the targeting complex, suggesting it is the site for target interaction. Since Met18 CT could not be purified, we instead used a crosslinking approach to identify protein‐protein interaction sites. Utilizing a DSS (disuccinimidyl suberate) crosslinker we observed the possible presence of crosslinked Met18 and Cia2 on an SDS‐Page gel. We are currently working towards verifying this through mass spectroscopy optimized for high molecular weight compounds. Additionally we are utilizing an in vivo sulfite reductase assay to monitor Met18 activity within living yeast. Our results have not only laid the foundation for further studies on Met18, but also jumpstarted the process of defining its functionality and understanding critical protein‐protein interactions responsible for target recognition and binding to CIA component, Cia2.
Protein‐Protein Interactions in Nickel Acquisition of Escherichia coli
Authors: Zeng Zhi Wei , Zamble Deborah
Published: November 2023

Nickel is a required cofactor for [NiFe]‐hydrogenase, an enzyme involved in bacterial metabolism and pathogenesis.1 Due to the toxicity of nickel,2 bacteria need to control its uptake and distribution intracellularly.3 In Escherichia coli, control of intracellular nickel is achieved by nickel uptake by membrane transporters and nickel delivery to the correct destinations by metallochaperone proteins; however, it is not clear how these two systems are linked. Previous results suggested that there is an interaction between the nickel metallochaperone HypB and a nucleotide binding domain of Nik transporter NikE. This protein‐protein interaction would support the hypothesis that HypB, a key protein in nickel delivery to the [NiFe]‐hydrogenase precursor protein, receives nickel directly from the Nik transporter. In the present work, the NikE‐HypB interaction is examined under various nucleotide‐loaded and metal‐loaded states of HypB.

Support or Funding Information

This work was supported in part by funding from NSERC and CIHR.
Secondary Structure Analysis of the C‐terminus of Ga‐interacting Vesicle Associated Protein Using Circular Dichroism Spectroscopy
Authors: Maddox Adam L , Bhandari Deepali
Published: November 2023

A vast majority of cell signaling is mediated through activation of hetero‐trimeric G proteins. Ga‐Interacting Vesicle associated protein (GIV) is a non‐receptor Guanine nucleotide exchange factor (GEF), which activates Gi family of heterotrimeric G proteins downstream of activated receptor tyrosine kinases (RTKs). GIV's GEF function is mediated by a stretch of highly conserved ~20 residues, which is followed by a putative SH2‐like domain in the C‐terminus of the protein. Previous studies have shown that the C‐terminal 211 amino acids of GIV (referred to as “GIV‐CT” henceforth) are capable of functioning autonomously from its recruitment to the cell surface upon activation of RTKs to promoting downstream signals by binding to and activating Gi proteins. However, despite all the functional information and computational predictions, the structural insights into how GIV‐CT is able to perform all these functions is missing. Here, we have attempted to probe the structural aspects of GIV‐CT using circular dichroism (CD) spectroscopy ‐ the most commonly used method for determining the secondary structure of peptides and proteins. N‐terminally His6‐tagged GIV CT wild type (WT) and a phosphomimetic mutant (S1674D; binds and activates Gai better than the WT) were expressed and purified from E. coli BL21‐DE3 cells using Co2+‐NTA affinity chromatography followed by cation exchange chromatography. Our preliminary CD spectroscopy analyses showed a random coil profile for GIV CT WT as well as S1674D mutant, both of which could be induced to adopt an a‐helical conformation by addition of trifluoroethanol (TFE). Further analysis of CD spectra to predict secondary structure characteristics was carried out using DichroWeb, an online deconvolution program, which compares CD data for known protein structure to that of unknown protein structure. Wild type GIV CT secondary structure was predicted to contain 1% α‐helix, 18% β‐sheet and 46% random coil while our mutant contained 1% α‐helix, 15% β‐sheet and 56% random coil. B‐turn and ‘denatured’ helix and sheet percentages make up the final total of 100% secondary structure. In the presence of TFE, GIV CT WT and 74D α‐helical content increased to 24% and 28%, respectively. Together, our data suggest that although recombinant GIV‐CT may be predominantly in an unstructured state, it likely has a propensity to fold into a regular secondary structure, perhaps upon interacting with a binding partner.

Support or Funding Information

This project is supported in part by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R25GM071638.
Structural Characterization of SOD4 from Candida albicans
Authors: Randolph Keyera , Galaleldeen Ahmad
Published: November 2023

Candida albicans is a fungal pathogen that infects immunocompromised individuals. During infection, the host's innate immune system responds by releasing reactive oxygen species (ROS) to fight off the invading microbes, however, the fungal pathogen is able to counter this response by releasing superoxide dismutase (SOD). SOD is an enzyme that catalyzes dismutation of superoxide radicals into hydrogen peroxide and molecular oxygen. There are several fungal SODs that play a vital role in defending the fungus against ROS such as Cu/Zn SOD (SOD1), SOD4, SOD5 and SOD6. The structure of SOD5 has been recently determined. It is similar to the dimeric SOD1 in that it maintains the beta‐barrel and the copper binding, but it lacks both the zinc‐binding site and the electrostatic loop, and it is monomeric. SOD4 is homologous to SOD5 and is predicted to be extracellular. SOD4 was overexpressed, and since SOD4 is insoluble, the protein was denatured, refolded and purified. Crystallization trials are underway in pursuit of the SOD4 structure.
Evaluating the role of a multi‐heme cytochrome c in electron transfer from an electrode surface to Heliobacterium modesticaldum
Authors: Herrera‐Theut Kathryn Ann , Gisriel Christopher , Laureanti Joseph , Orf Gregory , Baker Patricia , Jones Anne Katherine , Redding Kevin
Published: November 2023

Heliobacterium modesticaldum (H. modesticaldum) is an anaerobic photoheterotroph that can produce H2 when other electron transfer pathways are restricted. In addition, electrons can be injected into this organism via an electrode and redox mediator in a light‐dependent fashion, as shown recently by the Redding and Jones research groups. These factors make H. modesticaldum an ideal organism for use in a microbial photoelectrosynthesis cell, in which electricity can be used to power specific metabolic processes that produce a desired compound (e.g. H2). However, the injection of electrons into this organism has not yet been optimized. There is a gene (HM1_0653) in the genome encoding a multi‐heme cytochrome c that is similar to the proteins known to be used for exit of electrons in the well‐known electrode‐respiring bacteria (e.g. Geobacteria). RNA‐sequencing in the Redding lab has shown that the HM1_0653 gene is very poorly expressed in H. modesticaldum. Boosting expression of this cytochrome could lead to faster electron transfer into the cells and thereby more H2 production. This hypothesis is being tested by cloning the HM1_0653 gene into a plasmid behind a strong promoter and introducing it into H. modesticaldum using a transformation system recently developed in the Redding lab. To test that the protein is being highly expressed, SDS‐PAGE/heme‐staining will be conducted. The effect of HM1_0653 on electron transfer will then be assessed by comparing the light‐dependent current into cells containing the overproducing plasmid with control cells, as well as H2 production.

Support or Funding Information

I would like to thank Arizona Power Authority and the Arizona State University College of Liberal Arts and Sciences Scholarship for their support.
Conformational landscape of the protein kinase A Inhibitor PKI studied by fluorescence and NMR spectroscopy
Authors: Olivieri Cristina , Li Geoffrey , Kim Jonggul , Porcelli Fernando , Neibergall Matthew , Muretta Joseph M. , Thomas David D. , Taylor Susan S. , Gianluigi Veglia
Published: November 2023

Cyclic AMP‐Dependent protein kinase A (PKA) is a ubiquitous phosphoryl transferase that participates in numerous cellular signaling pathway. Inside the cell, the activity and localization of PKA are finely regulated by endogenous inhibitor proteins, such as the heat‐stable protein kinase A inhibitor (PKI). PKI plays a key role also in the nuclear regulation and cytoplasmic translocation of the catalytic subunit of PKA (PKA‐C). To understand the molecular mechanism underlying recognition of PKA‐C by PKI, we characterized the change in the conformational landscape of PKIα upon binding PKA‐C as well as the mechanism of binding, using solution NMR and fluorescence spectroscopy. We demonstrated that PKI free in solution has minimal secondary structural elements with only two transient helices and has transient intramolecular interaction between the C‐ and N‐terminus. When a ternary complex between PKA‐C, PKI and a non‐hydrolysable ATP mimic (ATPγN) is formed, only the portion of the peptide that directly interacts with the PKA‐C undergoes significant structural rearrangement, while the majority of the peptide is still unfolded. However, our experiments also indicated that binding to PKA‐C confers allosteric changes toward the nuclear export signal (NES) region present on PKI. Also our transient kinetics data reveal that the mechanism involves at least two distinct populations of PKA‐C binding to PKI with subsequent structural rearrangement and the flux through these competing pathways depend on ligand concentration. These studies not only elucidate the structural and mechanistic basis of PKA‐C:PKI interaction, but also represent the basis for further investigation on the role of the PKA‐C:PKI interaction toward regulation and nuclear export.

Support or Funding Information

NIH GRANT R01 from Gianluigi Veglia GM100301‐05
Crystal Structures of a Bacteriophytochrome from Myxobacteria Stigmatella aurantiaca
Authors: Waltz Patricia M , Halavaty Andrei , Patel Hardik , Hopkins James , Woitowich Nicole , Stojkovic Emina
Published: November 2023

Bacteriophytochromes (BphPs) are red‐light photoreceptors that regulate light responses in bacteria via reversible photoconversion between red (Pr) and far‐red (Pfr) light‐absorbing states. BphPs consist of a photosensory module that is composed of the PAS (Per‐ARNT‐Sim), GAF (cGMP phosphodiesterase/adenyl cyclase/FhlA), and PHY (phytochrome) domains. PHY domain is covalently linked to a signaling‐effector module such as histidine kinase. Wild type protein from non‐photosynthetic myxobacterium Stigmatella aurantiaca (SaBphP1) was purified, crystallized, and the structure of the complete photosensory module was determined at 2.35 Å resolution. Unlike classical BphPs, SaBphP1 undergoes limited Pr/Pfr photoconversion in the presence of the PHY domain. Wild‐type SaBphP1 lacks a conserved His that forms a hydrogen bond with the chromophore in the Pr state in the classical BphPs. A single Thr289His mutation in the chromophore‐binding pocket of SaBphP1 results in a complete Pr/Pfr photoconversion. Consequently, we determined the crystal structure of the complete photosensory module for SaBphP1 Thr289His mutant at 2.85 Å resolution. Both SaBphP1 crystal structures, solved in the Pr state, reveal how interdomain interactions bring together PAS, GAF and PHY domains to achieve Pr/Pfr photoconversion. We identify key amino acids that stabilize the chromophore in the Pr state and provide structural evidence to support the essential role of the PHY domain in Pr/Pfr photoconversion. Our results highlight local structural changes from the photosensory domain that significantly affect the long, signaling helices at the dimer interface and may impact the spatially distant histidine kinase domain and its activity.

Support or Funding Information

NSF MCB RUI 1413360
Evaluation of a Prefusion Model of the Herpesvirus Fusion Protein via Mutagenesis
Authors: Robinson Carolyn A , Gallagher Shannon L , Connolly Sarah A
Published: November 2023

Herpesviruses affect a significant portion of the human population and cause a variety of diseases, including cold sores, chickenpox, mononucleosis, and some forms of cancer. The first step of any viral infection requires a virus to enter a host cell. Herpesviruses enter cells by fusing a lipid viral envelope with the host cell membrane. Glycoprotein B (gB), a virally‐encoded class III fusion protein, mediates this fusion event by physically inserting into the host cell membrane and refolding from a prefusion to a postfusion conformation. The crystal structure of gB in its postfusion form has been solved, but its prefusion structure is unknown. A model of the prefusion gB structure was created based on homology to another class III fusion protein. To assess this model, we introduced mutations into gB that we predicted would affect its fusion capacity by altering the stability of its prefusion conformation. Mutations were designed to increase the flexibility of a coiled‐coil that is extended in the postfusion structure but broken in the prefusion model. gB is an essential protein that is conserved in all herpesviruses. Insights into the molecular details of how the gB protein refolds to force membranes to fuse may identify targets for antiviral intervention.

Support or Funding Information

DePaul University Research Council Grant
Investigation of the Molecular Basis of pH‐induced DNA Binding of CooA, a CO‐Sensing Transcription Factor
Authors: Weaver Brian , Westerlund Geoffrey , McGhiey Celia , Markovic Sam , Clark Robert
Published: November 2023

The carbon monoxide (CO) sensing heme protein, CooA, is a transcription factor that occurs in several bacteria and regulates the expression of genes, enabling these organisms to utilize CO as a sole energy source. Previous studies by our lab yielded the unexpected result that Fe(III) CooA binds DNA tightly at pH < 7, which deviates from the traditional understanding that CooA DNA binding is initiated only when the exogenous CO effector reacts with the Fe(II) CooA heme. This observation suggests that the disruption of one or more salt bridges upon effector binding may be a critical feature of the normal CooA activation mechanism. To test this possibility, a number of protein variants that eliminate selected salt bridges for the CooA homologs from Rhodospirillum rubrum and Carboxydothermus hydrogenoformans were prepared via site‐directed mutagenesis. Purified samples of these variant proteins, which were overexpressed in Escherichia coli, were then characterized by spectroscopic methods and functional assays to investigate the impact these mutations had on CooA heme coordination structure and DNA‐binding activity. Results of this work are presented in light of the accepted CooA activation mechanism.
NMR Structural Studies of Stress Responsive Peptide‐2 from the Insect Manduca sexta
Authors: Schrag Lynn , Cao Xiaolong , Herrera Alvaro I , Wang Yang , Jiang Haobo , Prakash Om
Published: November 2023

With the genome sequence of Manduca sexta available, we have identified ten open reading frames coding for precursors of paralytic peptide (PP) homologs: uENF1, uENF2, and SRP1–8. While uENF1 and uENF2 are located upstream of the PP gene, we temporarily call the other eight stress responsive peptides (SRPs), since stress is broader than infection, a type of biotic stresses. SRP2 is predicted to be a 25‐residue peptide (FGVKDGKCPSGRVRRLGICVPDDDY) stabilized with a disulfide bond. The proteolytic activation of proSRP2 is likely linked to the serine protease network that mediates and coordinates immune responses in this insect. SPR2 may also participates as a regulator in brain development. We have determined the solution structure of SRP2 by two‐dimensional 1H NMR spectroscopy to begin to understand structural‐functional relationships of this peptide. Our preliminary studies indicate that SRP2 has an ordered structure, which is composed of two short β‐strands at residues Arg12‐ Arg15 and Ileu18‐Val20, one type I′ β‐turns at residues Arg15‐Ileu18 and a γ′‐turn at residues Cys8 ‐ Ser10. Work is in progress towards 3D structure determination. Based on our NMR data, a well‐defined secondary and tertiary structure for this class of peptides will be presented.
The Role of a Highly Conserved Loop in Response Regulator Activation in the General Stress Response
Authors: Eaton Daniel S , Luebke Justin L , Crosson Sean
Published: November 2023

Bacteria depend on a diverse array of signaling proteins to sense and respond to their environments. An important class of proteins involved in such signaling cascades are the response regulators, which are controlled by the phosphorylation state of a conserved aspartate residue. Though most response regulators share great structural similarity, the allosteric pathway that governs their activation upon phosphorylation tends to vary. In this study, we examine the response regulator PhyR, a two‐domain protein comprised of a phohsphorylatable receiver domain and a σ‐like domain. This protein functions as an anti‐anti‐σ factor that governs activation of the general stress response (GSR) in alphaproteobacteria. Activated PhyR accomplishes GSR induction by binding to its cognate anti‐σ factor, NepR, through its σ‐like domain. This domain is occluded by the receiver domain in the inactive state. As such, in order for PhyR to carry out its binding activity, its receiver domain must undergo a significant conformational change to detach from the σ‐like domain. While a number of structural studies have focused on PhyR, a full characterization of the active state of the protein remains elusive. In the absence of definitive structural information about the protein's active state, we probed PhyR's activation mechanism through molecular dynamics (MD) simulations. From these simulations, we predicted that, for the protein to activate, a highly conserved loop in the receiver domain must be restructured in a manner which would disrupt a set of strong hydrogen bonds. Mutating the PhyR residues involved in these interactions results in hyperactive binding to NepR. Thus, we show that disruption of a particular interaction interface is sufficient to bias PhyR into the active state and therefore these residues play an important role in its activation dynamics.

Support or Funding Information

Funding: NIH 5R01Al107159
The B domain of the mitochondrial fission mechanoenzyme Drp1 is intrinsically disordered and coacervates under crowding conditions
Authors: Hill Blake , Posey Ammon , Bagheri Mehran , Kennedy Nolan , Khan Misha , Harden James
Published: November 2023

The Dynamin superfamily is a class of large GTPases that perform essential membrane remodeling events via self‐assembly stimulated GTP hydrolysis in a manner that involves the coordination of lipid‐membrane interaction, high‐order oligomerization, and GTP hydrolysis, however a clear understanding of how these functions are coordinated has remained elusive. A “variable domain” (VD) is present in many superfamily members and may enable specific targeting to sites of action, but the nature of VD function is unclear in most dynamins. Dynamin‐related protein 1 (Drp1) is the primary mechanoenzyme responsible for mitochondrial fission. We seek to identify the role of the Drp1 VD, or B domain. We find that removal of the B domain from Drp1 results in enhanced assembly and GTP hydrolysis, suggesting that the B domain has an auto‐inhibitory role. We find that the B domain is intrinsically disordered (ID) and surprisingly, undergoes phase separation or coacervation under conditions that induce other ID proteins to fold. The same conditions that induce phase separation of the B domain also enhance binding to cardiolipin, a lipid that is unique to the mitochondrial membrane. Based on these findings we suggest a model in which the B domain inhibit Drp1 assembly by dynamically occluding assembly interfaces, and that this auto‐inhibition is relieved upon interaction of the B domain with cardiolipin at the mitochondrial membrane, involving the process of coacervation. This model may also be applicable to other members of the dynamin superfamily, and intrinsically disordered membrane remodeling proteins in general.

Support or Funding Information

NIH R01‐GM067180
Regulatory role of non‐phosphorylated tau 441 and its antibodies on tubulin polymerization
Authors: Imhof Matthew M. , Zabik Nicole L. , Martic Sanela
Published: November 2023

Tau protein is a microtubule (MT) associated protein which functions to promote polymerization and stability of microtubules (MT) in neuronal cells. When tau undergoes post‐translational modification, it misfolds and aggregates, forming cytotoxic species which lead to neurodegeneration. Immunotherapies against tauopathies have shown promise in animal models by inducing clearance of tau pathology. However, the exact mechanisms of antibody‐based inhibition of neurodegeneration remain unidentified. We have reported on anti‐tau antibodies to non‐phosphorylated tau441 and their inhibition of tau aggregation in vitro. We also showed that phosphorylation of tau441 at Ser199, by GSK‐3β, may be inhibited by anti‐tau phosphospecific antibodies. Here, we have evaluated polymerization of tubulin in the presence of non‐phosphorylated tau441 and anti‐tau antibodies to non‐phosphorylated tau [targeting epitopes in the N‐terminal, R4, and C‐terminal]. ELISA was used to determine antigen‐antibody binding affinities between tau, tubulin and anti‐tau antibodies. An anti‐tubulin antibody was used as a control in the polymerization and ELISA experiments. Using the tubulin fluorescence polymerization assay, we have discovered that the antibodies specific to non‐phosphorylated tau441 reduced tubulin polymerization. In the presence of non‐phosphorylated tau441, we observed successful tubulin polymerization even in the presence of anti‐tau antibodies. These studies indicate that tau plays a regulatory/competitive role in MT formation

Support or Funding Information

NIH/NIGMS/R15GM11905301
High throughput quantification of nucleated protein phase behavior in vivo
Authors: Halfmann Randal , Khan Tarique , Bruner Ellen , Kandola Tejbir
Published: November 2023

Phase separation is a foundational principle for spatiotemporal regulation of protein activity. A subset of phase separating proteins, known as prions, produce switch‐like, irreversible changes in protein activity that effectively decide the fates of cells and those of their progeny. For some proteins, these switches are physiological, as we have recently shown for certain innate immune signaling proteins. But for others, they are clearly pathological, as for Alzheimer's and other degenerative disease‐associated proteins. What physical properties distinguish physiological from pathological prions, and how does prion formation differ from that of other condensed protein phases such as liquid droplets? In order to decipher the rules that govern prion‐like phase transitions and explore the breadth of their biological effects, we developed a powerful cell‐based fluorescence assay termed Amphifluoric FRET (AmFRET). AmFRET combines the use of photoconvertible fluorescent proteins with a high‐variance protein expression system and imaging flow cytometry to enable precise, high‐throughput mapping of the concentration‐dependencies and kinetics of protein self‐assembly. We have used AmFRET to characterize phase behaviors for hundreds of known and putative prion‐forming proteins from two extremes of conformational space: 1) intrinsically disordered regions commonly involved in gene regulation, and 2) well‐folded death domains from mammalian signaling pathways. Our findings enable us to derive a quantitative physical description of “prionness”, and reveal an unprecedented prevalence of prion behavior in cell signaling pathways that execute terminal cell‐fate decisions. In addition, we have discovered a multiplicity of nucleated phases accessible to individual proteins; the phases differ in their thermodynamic properties and biological consequences.

Support or Funding Information

This work is funded by an NIH Director's Early Independence Award (DP5OD009152) and the Stowers Institute for Medical Research.