introduction
what is it good for ?
• Biomolecular interactions are intrinsic and vital to many cellular processes
• Most of the work of living organisms is performed by proteins
• Proteins do their work by acting on other macromolecules: nucleic acids, carbohydrates, lipids, and, especially,other proteins
Biomolecular Interaction Analysis provides qualitativeand quantitative information. Basis for
understanding many biological processes
• Interaction Trap/Two-Hybrid System to Identify Interacting Proteins• High-Throughput Screening for Protein-Protein Interactions Using Yeast Two-Hybrid
Arrays• Phage-Based Expression Cloning to Identify Interacting Proteins• Detection of Protein-Protein Interactions by Coprecipitation• Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET)
Microscopy• Identification of Protein Interactions by Far Western Analysis• Scintillation Proximity Assay (SPA) Technology to Study Biomolecular Interactions• Identifying Protein Interactions by Hydroxyl-Radical Protein Footprinting• Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence
Complementation (BiFC) Analysis• Production and Use of Trimeric Isoleucine Zipper Fusion Proteins to Study Surface
Receptor Ligand Interactions• Fluorescence Quenching Methods to Study Lipid-Protein Interactions• Determination of Protein Contacts by Chemical Cross-Linking With EDC and Mass
Spectrometry• Membrane-Based Yeast Two-Hybrid System to Detect Protein Interactions• Detection and Analysis of Protein-Protein Interactions of Organellar and Prokaryotic
Proteomes by Blue Native and Colorless Native Gel Electrophoresis
introduction
qualitative methods
• Measuring Protein Interactions by Optical Biosensors
• Analytical Ultracentrifugation: Equilibrium Approach
• Analytical Ultracentrifugation: Sedimentation Velocity Analysis
• Titration Microcalorimetry
• Reduced-Scale Large-Zone Analytical Gel-Filtration Chromatography for Measurement of Protein Association Equilibria
• Size-Exclusion Chromatography with On-Line Light Scattering
• Spectroscopic Methods for the Determination of Protein Interactions
• Application of Amide Proton Exchange Mass Spectrometry for the Study of Protein-Protein Interactions
• Circular Dichroism to Study Protein Interactions
• Quantitative Determination of Protein Stability and Ligand Binding by Pulse Proteolysis
introduction
quantitative methods
introduction
http://onlinelibrary.wiley.com/book/10.1002/0471140864
Wiley Online Library Current Protocols in Protein Science
http://www.els.net/
Encyclopedia of Life Sciences
• The strength of an noncovalent interaction between two molecules is described by equilibrium parameters
A + B [AB]
equilibrium dissociation constant: Kd = [A][B]/[AB] = koff /kon
where [AB] is the concentration of the complexed species and [A] and [B] are the concentrations of the noncomplexed species. Concentrations are given in molar terms, as is the Kd
basic concepts
how does it work ?
kon
koff
a low Kd corresponds toa high „affinity“
or better, „binding strength“
• Quick yes or no answer
• SPR range for affinity (Kd) measurement: 10pM to 1mM
• Affinity maturation
• Comparison of different mutants/constructs
• Full kinetic and thermodynamic characterisation (conf. state
trapping, comparison MD/NMR/kinetics)
• Confirmation of affinities obtained by other methods
• Epitope mapping
• Concentration measurements
• Quality control
introduction
SPR / what is it good for ?
basic concepts
measuring principle
• SPR detects refractive index changes close to the surface (0 – 100nm)
• accumulation of 1 pg substance/mm2 gives a change of 1 µRIU or 1 RU
basic concepts
correlation of SPR response with absolute surface protein concentration
• signal proportional to mass
• same specific response for different proteins
Protein
Chymotrypsinogen ATransferrinanti-Transferrinanti-β2microglobulin
MW
2570084000150000150000
Symbol
▲
dR/dt = kon ∙ C ∙ [Rmax - R] - koff ∙ R
RU/s M-1s-1 M RU s-1 RU
d[AB]/dt = kon ∙ [A] ∙ [B] - koff ∙ [AB]
basic concepts
information content of a sensogram
kon
koff
A + B [AB]
• All four compounds have the same affinity KD = 10 nM = 10-8M
• The same affinity can be the result from different kinetics !
time
RU
10-5103
10-4104
10-3105
10-2106
kd
[s-1]
ka
[M-1s-1]
KD 10 nM
basic concepts
kinetic resolution of affinities with SPR
basic concepts
kinetic resolution of affinities with SPR
koff (s-1)
k on
(M-1
s-1 )
104
0.0001 0.001 0.01 0.1 1
107
106
105
102
103
1 nM100 pM 10 nM10 pM
100 nM
1 µM
1 mM
100 µM
10 µM
KD
• HIV-1 protease inhibitors: on-off rate map
koff (s-1)
k on
(M-1
s-1 )
104
0.0001 0.001 0.01 0.1 1
107
106
105
102
103
1 nM100 pM 10 nM10 pM
100 nM
1 µM
1 mM
100 µM
10 µM
KD
B435
B408
B347
B365
B425
B439
A016
B249
B277
A018
A015
A017
B322
B440
B429A03
7
B409
B388
B268
A038
B412
B295B355
B369
Nelf
RitSaq
Amp
U75875
Ata
B268
basic concepts
kinetic resolution of affinities with SPR
• HIV-1 protease inhibitors: on-off rate mapDevelopment of Saquinavir, Amprenavir and Atazanavir
investigating biomolecular interactions with SPR sensors
applications
• Protein – Protein Interactions
− recombinant mouse prion protein – IgG POM1
− L. lactis multidrug exporter LmrCD – DARPins
− arginyl-tRNA synthetase – ribosomal protein L3
− human Nogo-A – Nogo-A receptor
− human IL18 and IL33 – IL18BP
− PhyR – NepR
− p150 – hMSH6
− SicP/SptP complex – ATPase InvC
− AcrB (multidrug efflux pump membrane protein) – DARPins
− CitS (transport membrane protein) – DARPins
− Caspase-1, -2, -3, -5 and -8 (Proteases in Apoptosis) – DARPins
− Caspase-1, -2, -3, -5 and -8 – PRYSPRY (C-terminal domain of Pyrin)
investigating biomolecular interactions with SPR sensors
applications
• Protein – Protein Interactions
− HLA B27 – Fab (reagent to investigate dimerization, impact on Bechterew's disease)
− rhCAIX – Fab (tumor targeting)
− rhFAP (fibroblast activating protein) – aFAPhGITR-L (ligand for GITR on T cells)
− Nimak – HcpC
− HIV Capsid Protein – PRYSPRY (C-terminal domain of antiviral factor TRIM5α)
− Cellular receptor CD46 – Ad3 (7,11,35) - Fiber Knobs
− VEGF – VEGF receptor
− MSG1 – Actin
− chaperone SicP – effector protein SptP
− S.aureus Orf1055 – unknown protein interaction partner (ligand fishing)
investigating biomolecular interactions with SPR sensors
applications
• Protein – Peptide Interactions
− prion protein PrP – amyloid-β− single chain Fv – biotinylated-nitrotyrosine containing 10-14mers− anti-PrP monoclonal antibodies – PrP peptide set (12mers)
• Protein – Small Molecule Interactions
− M. tuberculosis secreted chorismate mutase – transition state analogon− polyclonal IgGs – polythiophene acetic acid− cell wall binding domain protein CBDP35 – N-acetyl-D-glucosamine− M. tuberculosis IspF – thiazolopyrimidines− hCarbonic Anhydrase Ι – sulfonamide inhibitors
• Protein – RNA Interactions
− SerRS – seryl-tRNA
investigating biomolecular interactions with SPR sensors
applications
• Protein – Carbohydrate Interactions
− Influenza A Virus H5N1 hemagglutinin – synthetic trimeric sialylglycan− Nkp30 – synthetic heparin oligosaccharides− Moesin – Glycosylphosphatidylinositol
• Protein – Lipid Interactions
− ApoM – lauryl-oleyl-phosphatidylethanolamine
• Protein – Cell Interactions
− cell adhesion protein fasciclin – whole M. extorquens cells
• Large RNA – Small Molecule Interactions
− E.coli btuB riboswitch (205nt) – adenosyl-cobalamine
• DNA – DNA interactions
− Biotin-TTTCCTCAGCATCTTATCCGAGTTT – ATGCTGAGG (and variants)
applications
example: E.coli btuB riboswitch – cobalamine (vitamin B12)
5`- untranslated region of btuB transcript
R =
vitamin B12
C72H100CoN18O17P 1579,60 g/mole
P. Choudhary / Sigel Group / UZH
• Surface plasmon resonance detects binding events as changes in refractive index in the volume
between 0 and 100 nm from the chip surface
• Real-time kinetic measurements
• No labeling of interactants necessary
• Low sample amounts (micrograms) required
• High Throughput SPR needs are covered by BioRad`s ProteOnXPR36
summary
• importance of sample preparation:
1. purity
2. homogeneity
3. stability
• further reading on surface plasmon resonance (SPR):
Surface Plasmon Resonance Based Sensors, Homola, Springer 2006
take home message