# Presentation: 376168

## Protein Chemistry Laboratory

- Robert J. Fisher
- Cancer Biotechnology
- April 5, 2007

## PCL Capabilities

- Protein Characterization
- Edman amino acid sequencing
- HPLC purifications of proteins and peptides
- Maldi-Tof mass spectrometry
- Molecular Interactions
- SPR Spectroscopy
- Fluorescence anisotropy
- Fluorescence life-time measurements

## Organization of the PCL

- Protein Chemistry (301-846-6745)
- 		Oleg Chertov, Ph.D. and Young Kim
- Maldi-Tof Mass Spectrometry (301-846-6702) 	John Simpson, Ph. D.
- Characterization of Ligand Interaction (301-846-1634)
- 		Andrew Stephen, Ph.D. Karen Worthy, MS and Lakshman Bindu, MS

## Protein Chemistry Techniques and Core Activities

- Complete characterization of a protein
- Non-routine protein identification
- Purification of peptides, proteins and labeled oligonucleotides
- Identification of cross-linked amino acids, post-translational modifications including phosphorylation
- High sensitivity Edman amino acid sequencing
- MALDI-TOF mass spectrometry

## Why do Edman Amino Acid Sequencing?

- Best method to verify the amino terminus of  a protein
- Quantitative technique -  PTH amino acid analysis by HPLC is precise and quantitative
- Important in the Quality Control ie ‘Total Characterization’ of a purified or recombinant protein

## Edman Amino Acid Sequencing

- coupling

- cleavage

- Conversion to
- PTH Amino Acid

***Edman Amino Acid Sequencing***

## Complete Protein Characterization 

## Topics:

- How does BIAcore work?
- microfluidics
- S*olid Phase ligand binding*
*Surface Plasmon Resonance*
- What can BIAcore do?
*Realtime Binding Kinetics (on and off rates)*
*Concentration measurement**	*
*Equilibrium constant*
*Data analysis**	**	**	**.*

## Basic Principle

- A binding molecule is bound to the sensor surface.(ligand –peptide, protein, sugar, oligonucleotide))
- Another  *(the analyte)* is passed over the surface and binds to it.

## Experimental Design

## Sensor Chip CM-5: Carboxymethylated dextran coated surface.

** ****Allows covalent coupling via -NH****2****, -SH, and -CHO **

***Sensor Chip CM-5:****** ******Carboxymethylated dextran coated surface.***

## The Flow Cell

- Surface is divided into 4 channels, which can be used individually or in a number of combinations

## Microfluidic System

- Low reagents consumption
- Efficient mass transport
- Low dispersion
- Highly reproducible injections; CV typically less than 1%
- Wide range of contact times, 1 s - 12 h
- Sample recovery and fractionation

## Measurement of Binding

- Binding is measured as a change in the refractive index at the surface of the sensor
- This is due to ‘Surface Plasmon Resonance’ (SPR)
- The change in refractive index is essentially the same for a given mass concentration change *(allows mass/concentration deductions to be made)*
- Binding events are measured in real time  *(allowing separate on and off rates to be measured.)*

## Theoretical Considerations

- Binding is measured as a change in the refractive index at the surface of the sensor...

**How?**

## Total Internal Reflection

***Total Internal Reflection***

- Although no photons exit the reflecting surface, their electric field extends ~1/4 wavelength beyond the surface.

## Resonance Surface Plasmon 

- If a thin gold film is placed on the reflecting surface, the photons can interact with free electrons in the gold surface.

- Under the right conditions, this causes the photons to be converted into plasmons and the light is no longer reflected.

## Surface Plasmon Resonance

- This occurs when the *incident* light vector is equal to the surface plasmon vector....

## Effect of binding on SPR

- This is affected by changes in the medium (eg binding of analyte), and results in a change in the velocity of the plasmons.
- This change in velocity alters the incident light vector required for SPR and minimum reflection.

***Effect of binding on SPR***

## How does BIACore Measure this? 

- Fixed wavelength light, in a fan-shaped form, is directed at the sensor surface and binding events are detected as changes in the particular angle where SPR creates extinction of light.

## The Sensorgram

**Notes:**
The progress of an interaction is monitored as a sensorgram. Analyte binds to the surface-attached ligand during sample injection, resulting in an increase in signal. At the end of the injection, the sample is replaced by a continuous flow of buffer and the decrease in signal now reflects dissociation of interactant from the surface-bound complex. A response of 1000 RU corresponds to a change in surface concentration of 1 ng/mm2.

## Surface Plasmon Resonance

- time

- response

## Binding Analysis

- How Much?**	**

*Active Concentration*

- Kinetics

*Affinity*

*Specificity*

- How Fast?**	****	**

- How Strong?

- How Specific?**	**

## Concentration

- Signal proportional to mass

- Same specific response for different proteins

## Topics 

- HIV nucleocapsid binding to short oligonucleotides
- HIV gag precursor protein binding to short oligonucleotides
- Antibody screening
- Affibody Characterization
- Small molecule binding to SH2 domains

## HIV NC and Gag Precursor to Short Oligonucleotides

**Alan Rein**
- HIV Drug Resistance Program, CCR

## Why study HIV NC interactions with Oligonucleotides?

- NC is highly conserved
- NC alone or as part of Gag is involved in many stages of the HIV life cycle
- NC is a nucleic acid chaperone
- A static binding model is not sufficient to explain all of NC’s activities
- Minimal step in HIV particle assembly
- Insights into binding of gag precursor

## Nucleocapsid is a domain of the Gag polyprotein

**p6**

**MA**

**CA**

**NC**

**Gag**

**Proteolysis**

## HIV NC binding to short oligonucleotides

***HIV NC binding to short oligonucleotides***

**d(TG)****4 ****ligand**

**d(TG)4 ligand**

**d(TG)4 ligand**

**d(A)****8**** ligand**

**250mM NaCl**

## Working Model for NC Binding to Short Oligonucleotides

## Complex binding model – Matt Fivash, DMS

***Complex binding model – ******Matt Fivash, DMS***

- 	N = 	NC
- 	O = 	oligonucleotide
- 	NO = 	1:1 complex
- 	NON = 	2N bound to oligonucleotide
- 	ONO = 2 oligonucleotides bound to NC

## Intrinsic tryptophan quenching by oligonucleotide

***Intrinsic tryptophan quenching ******by oligonucleotide***

**Oligonucleotide titrated into HIV NC (excess NC)**

**NON**

**NO**

**ONO**

## Evidence to Support Model

- SPR spectroscopy—Direct binding experiments (Evidence for NO and ternary complexes NON, ONO)
- Tryptophan quenching (Evidence for NO and ternary complexes NON, ONO)
- Fluorescence  Anisotropy (Evidence for NO and higher order complexes)
- The N-terminus of NC and intact zinc fingers are required for high affinity binding
- FTIR ESI Mass spectrometry identified NO and NON complexes

## HIV-1 Gag - Basics

- Expression of a single viral protein (Gag) in mammalian cells, is sufficient for assembly of virus-like particles
- Gag specifically packages genomic RNA during virus assembly
- Gag can use genomic RNA or cellular mRNA as “scaffolding” during virus assembly
- Gag is responsible for annealing of nucleic acids at several stages of the viral replication cycle

## Assembly of HIV-1 virus-like particles in vitro

**Assembled particle**

**HIV Gag precursor protein**

- +  **RNA**

- 	HIV-1 Gag (missing the p6 domain) when incubated with **nucleic acid** can assemble into virus-like particles
- 	(Campbell and Rein, J. Virol. [1999] **73:** 2270)

## Eleven replicate injections of NC (200nM) used to calibrate the density of a TGx10 Biacore chip

## Kinetic binding of Gag (0.19 – 400nM) to 0.36 RU’s of TGx10

## Binding Site Analysis

***Binding Site Analysis***

## Conclusions

- ESI-FTMS data suggest the binding site for NC is 5 bases.
- NC injections can be used to calibrate the surface density of ultra-low oligo surfaces.
- Gag binds with high affinity to TGx10 oligos .
- Steady-state binding can be fit with a 2-site binding model (Kd1 1.5 nM, Kd2 160 nM).
- The binding of Gag to TGx10 does not saturate even at stoichiometries of  >6 Gag molecules/TGx10.
- These data suggest that once Gag has filled the TGx10, additional Gag can bind, forming TGx10-Gag-Gag complexes.

## Kinetic Screening of Monoclonal Antibodies

**Ira Pastan**
- Laboratory of Molecular Biology, CCR

## How does kinetic screening work?

- Rabbit antimouse C domain polyclonal antibodies are co-valently attached to a CM-5 sensor chip.
- A 1:1 dilution of hybridoma supernatant is passed over the ramC, capturing 200-500 RU’s of MAb.  This process repeated on two other surfaces and the fourth is a control.
- An appropriate concentration of antigen is serially injected over all 4 flowcells, followed by surface regeneration.

## Arrangement of surfaces in a CM5 sensor chip utilizing Serial Flow Cell

***Arrangement of surfaces in a CM5 sensor chip utilizing Serial Flow Cell***

**Sample in**

**Sample out**

**RAMC Control FC1**

**RAMC MAb1 FC2**

**RAMC MAb2 FC3**

**RAMC MAb2 FC4**

## Pastan MAbs 1-60 blank referenced

***Pastan MAbs 1-60 blank referenced***

## Three distinct patterns observed

- Typical binding curve-although some individual MAbs have distinctly slower off rates (MAb47)
- Unusually low response
- Complex Binding

## MAb47 

**Ka=1.02e5/Ms **
**kd=1.1e-7/s**
**KD=1.08e-12M**

## MAb35

**Ka=5.0e4/Ms **
**kd=2.2e-3/s**
**KD=4.3e-8M**

## MAb12

- Simple Model does not fit data

- A+B>AB

## MAb12 – Complex Model

**A+B>AB**
**A+C>AC**

**ka1=1.8e5/Ms **
**kd1=1.2e-4/s**
**KD1=6.4e-10M**

**ka2=8.5e5/Ms **
**kd2=3.7e-3/s**
**KD2=4.3e-9M**

## Rapid Screening of Crude Hybridoma Supernatants

- 60 MAbs screened using two Biacore® 2000 instruments in an overnight experiment
- Possible to obtain MAb concentration, kon and koff rates
- Rapid qualitative assessment of MAb quality
- Biacore ® A100 would allow a fivefold increase in throughput
- Biorad ProteoN XPR 36

## Biacore A100 Chip

***Biacore A100 Chip***

## Rapid screen of crude hybridomas - Assay setup Biacore A100

- (1 of 4 flow cells shown)

- 300 samples tested against antigen for binding and to provide dissociation rate ranking
- mAb concentrations in hybridoma samples unknown
- Immobilizing different levels of capture Ab on each spot gives a range of mAb-binding capacities.
- Maximum information extracted with this strategy

- mAb (mIgG) from hybridoma

- Antigen

- CM5

- Capture Ab - Rabbit anti-mIgG(fc)

**Notes:**
Note that this is a different setup to that used by Abgenix. Here, GlaxoSmithKline employ a lower sample throughput approach (1 hybridoma per cycle as opposed to 4/cycle) that aims to extract more information for each mAb screened and properly evaluate the setup prior to detailed kinetic assays.

The ”maximum information” refered to in the last bullet refers both to the binding characteristics of each mAb screened, and in terms of the assay design itself.

## Ultra-low Analyte Concentrations Affibody against Her/Erb2

**Jacek Capala **
- Radiation Oncology Branch, NIH/NCI

## Targeting Molecules: Affibody® 

***Targeting Molecules: Affibody******®****** ***

## Targeting Molecules: Affibody®

***Targeting Molecules: Affibody******®***

## Affibody® Molecule: Diversity

- Randomization of 13 selected positions

- Protein A-domain
- scaffold

- 3x109 Affibody®library members

***Affibody******® ******Molecule: Diversity***

## Experimental setup

- Rabbit Antimouse C (RamC) is covalently attached to each flow cell
- FC fusion protein her2/erb2 extracellular domain is captured onto flowcells 2 and 4
- An ultra low concentration affibody is injected serially over all four flowcells for 2000 seconds, this is followed by two injections of buffer, but NO regeneration step is used

## Titration of 7.8 to 500 picomoles of her2 z342 affibody on to captured her2/erb2 ECD

***Titration of 7.8 to 500 picomoles of her2 z342 affibody on to captured her2/erb2 ECD***

**Concatenated T100 Data**

**Unprocessed T100 Data**

**-10**

**0**

**10**

**20**

**30**

**40**

**50**

**60**

**70**

**Sensorgram**

**Response Units (RU's)**

**s**

**Time**

**Response Units (RU's)**

**-10**

**0**

**10**

**20**

**30**

**40**

**0**

**500**

**1000**

**1500**

**2000**

**2500**

**0**

**1000**

**2000**

**3000**

**4000**

**5000**

**Time**

**kt=5.5e****8****, k****on****=3.3e****6****, k****off****=9e****-5**** KD = 27.2 pM**

## Biacore T100 

- Latest version of high resolution SPR

## Biacore T100 Features

- Online buffer degassing (allows up to four buffers to be used in an experiment)
- Out gassing in a varying temperature experiment is not a problem
- Two pairs of parallel flow cells which do not require hydrodynamic addressing
- Optimized for thermodynamic experiments

## T100 experiments done at 8 different Temperatures

## Beta2microglubulin binding to mAB 5°  C

## Beta2microgolbulin binding to mABat 40 degrees C

## Slide 63

## 1KJ = 0.239005736 Kcal

| Parameter Name | Parameter Value | SE |
| --- | --- | --- |
| ΔH° [kJ/mol] | -40 | 1.5 |
| ΔS° [J/(K*mol)] | 11 | 5 |
| TΔS° [kJ/mol] | 3.2 | 1.5 |
| ΔG° [kJ/mol] | -44 | 0.016 |
| ΔCp° [kJ/(K*mol)] | 0.65 | 0.26 |
|  |  |  |
| ΔH°‡ ass. [kJ/mol] | 36 | 4.4 |
| ΔS°‡ ass. [J/(K*mol)] | -29 | 15 |
| TΔS°‡ ass. [kJ/mol] | -8.6 | 4.5 |
| ΔG°‡ ass. [kJ/mol] | 45 | 0.048 |
| Ea ass. [kJ/mol] | 38 | 4.4 |
|  |  |  |
| ΔH°‡ diss. [kJ/mol] | 78 | 3 |
| ΔS°‡ diss. [J/(K*mol)] | -33 | 10 |
| TΔS°‡ diss. [kJ/mol] | -9.8 | 3 |
| ΔG°‡ diss. [kJ/mol] | 88 | 0.032 |
| Ea diss. [kJ/mol] | 81 | 3 |

**1KJ = 0.239005736 Kcal**

## Sample and Time consumption

- Experiment took about 48 hours run and about one hour of data processing
- 3 micrograms of mAb against beta2microglubulin was amine coupled to spot 2
- 1ml  of a 85nM solution of beta2microglobulin was consumed (concentration series of 0.1 - 85nM) for entire experiment
- Need about 100 fold more reagents for ITC

## Peptide Binding to SHC-SH2 Domain

**Terrence Burke**
- Laboratory of Medicinal Chemistry, CCR

## SHC protein

- Non-catalytic SH2 domain-containing docking modules
- Associated with proliferation, survival and apoptosis
- Downstream signaling of receptor tyrosine kinases (RTKs)
- Link activation of the cytoplasmic kinase domains with Ras effectors
- Disruption of Shc-dependent signaling through blockade of its SH2 domain interactions may indicate a new therapeutic approach

## Phosphopeptide Binds Weakly to SHC Protein

**WJC-01 ****	****8.40 x10****-5****M****	****	****Ac-LpYQGLS-amide**
**WJC-02****	****1.00 x10****-2****M****	****	****Ac-LYQGLS-amide**

***Phosphopeptide Binds Weakly to SHC Protein***

**Blue****	****Phosphopeptide**
**Red****	****Nonphosphorylated**

## Fluorescence Anisotropy

- If a fluorescent molecule is excited with polarized light, the emission may also be polarized
- Molecule must contain a transition moment for absorption and emission within a defined orientation within the structure
- Homogeneous assay
- Fast – plate reader 384 well format
- Method of choice for HTS

## Protein binding using fluorescence anisotropy

**Peptide**

**Fluorescein**

**Peptide**

**Fluorescein**

**Polarized Excitation Light**

**Small peptide**

**Rapid Rotation**

**Emitted Light is Depolarized**

**Peptide**

**protein**

**Peptide**

**protein**

**Polarized Excitation Light**

**Large complex**

**Slower Rotation**

**Fluorescein**

**Fluorescein**

**Emitted Light remains Polarized**

**Notes:**
This is a cartoon showing how we measure the binding of a protein to a small oligonucleotide by fluorescence anisotropy. We have also measured the binding of peptides to proteins and small molecules to proteins.

## Fluorescence Anisotropy

**SHC Protein (Molar)**

**Milli-Polarization Units**

**0**

**50**

**100**

**150**

**200**

**250**

**0**

**2e-5**

**4e-5**

**6e-5**

**8e-5**

**1e-4**

**Column A**

**WJC-03**

## Eleven compounds tested in an afternoon

| Won-Jun Choi | KD | Peptide |
| --- | --- | --- |
| WJC-01 | 8.40E-05 | Ac-LpYQGLS-amide |
| WJC-02 | 1.00E-02 | Ac-LYQGLS-amide |
| WJC-03  | 3.15E-05 | FITC-pYQGLS-amide |
| WJC-05 | 3.86E-06 | FITC-NH-(CH2)2-CO-pYQGLS-amide |
| WJC-06 | 1.87E-05 | FITC-NH-(CH2)3-CO-pYQGLS-amide |
| WJC-08  | 1.16E-05 | FITC-NH-(CH2)5-CO-pYQGLS-amide |
| WJC-07  | 1.47E-06 | FITC-NH-(CH2)4-CO-pYQGLS-amide |
| WJC-11 | 1.25E-05 | FITC-NH-(CH2)2-CO-LpYQGLS-amide |
| WJC-12  | 1.13E-05 | FITC-NH-(CH2)3-CO-LpYQGLS-amide |
| WJC-13  | 9.12E-06 | FITC-NH-(CH2)5-CO-LpYQGLS-amide |
| WJC-16 | No Binding | FITC-NH-(CH2)4-CO-YQGLS-amide |

***Eleven compounds tested in an afternoon***

## Phospho-peptide shows high affinity binding in Biacore experiment 

## Screening of unlabeled compounds using fluorescence anisotropy 

## Conclusions FA experiments

- Position and linker length of fluorescein label are important for binding affinity for Shc
- No binding seen with non-phospho-peptide
- No binding to other SH2 domain protein
- SPR studies are consistent with fluorescence anisotropy results
- FA can be used to ‘screen’ unlabeled peptides

## Conclusions

- The PCL is positioned to measure molecular interactions of protein/nucleic acid, protein/protein and small molecule to protein ligand.
- The PCL has the highest resolution SPR instruments (noise is ± 0.2 RU’s at 10 Hz)
- The PCL has an evolving expertise in fluorescence spectroscopy
- The PCL has the highest sensitivity Edman sequencing (low picomolar), can purify anything from anything by HPLC and can develop Maldi-tof methods