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
Topics: How does BIAcore work? • microfluidics • Solid 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 • Direct coupling of Ligand to Surface. • Indirect, via a capture molecule (eg a specific IgG). • Membrane anchoring, where the interacting ligand is on the surface of a captured liposome.
Sensor Chip CM-5:Carboxymethylated dextran coated surface. Allows covalent coupling via -NH2, -SH, and -CHO
The Flow Cell F1 & 2 F1 F3 & 4 F2 F1 - 3 F3 F1 - 4 F4 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 At a certain angle of incidence, light entering a prism is totally internally reflected. (TIR). 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 • Plasmons create an electric field (evanescant) that extends into the medium surrounding the film • 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.
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.
Surface Plasmon Resonance Equilibrium, KD = kd / ka Association - ka Dissociation - kd buffer response time
Binding Analysis • How Much? Active Concentration • How Fast? Kinetics • How Strong? Affinity • How Specific? Specificity
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 Zn Zn + + + + + + + + + Gag Proteolysis MA CA NC p6
HIV NC binding to short oligonucleotides d(TG)4 ligand d(A)8 ligand d(TG)4 ligand d(TG)4 ligand 250mM NaCl
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 NON NO ONO Oligonucleotide titrated into HIV NC (excess NC)
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 MA MA CA MA MA MA CA MA CA NC NC NC MA MA CA MA MA MA NC CA NC MA CA RNA NC NC NC CA MA CA MA MA MA NC NC CA CA MA MA MA MA + RNA HIV Gag precursor protein Assembled particle • HIV-1 Gag (missing the p6 domain) when incubated with nucleic acid can assemble into virus-like particles • (Campbell and Rein, J. Virol.  73: 2270)
Eleven replicate injections of NC (200nM) used to calibrate the density of a TGx10 Biacore chip 3 2.5 2.0 2 1.5 1 Response Units (RU) 1.0 Response Units (RU) 0.5 0 0.0 -0.5 -1 20 40 60 80 100 -50 0 50 100 150 200 250 Injection Cycle Time (s)
Kinetic binding of Gag (0.19 – 400nM) to 0.36 RU’s of TGx10 25 8 Gag's bound 20 15 Response Units 4 Gag's bound 10 2 Gag's bound 5 1 Gag bound 0 -50 0 50 100 150 200 250 Time (s)
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 Sample in RAMC Control FC1 RAMC MAb1 FC2 RAMC MAb2 FC3 Sample out RAMC MAb2 FC4
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 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 A+B>AB A+C>AC
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