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125:583 Biointerfacial Characterization: Protein-Protein
InteractionsOctober 30, 2006
Gary Brewer, Ph.D.Dept. of Molecular Genetics, Microbiology & Immunology
UMDNJ-RWJMS
The flow of “omics” research
Genome↓
Transcriptome↓
Proteome↓
Interactome
The interactome
•Proteins rarely (if ever) act alone
•Components of biomolecular machines
•Estimate: average of 5 interacting partners per protein
•For examples of interactomes, see http://bond.unleashedinformatics.com/Action (Must register for free account)
Identification and characterization: A formidable
problem•Proteins have very diverse
physiochemical properties
•Equilibrium dissociation constants can vary over several orders of magnitude
•Proteins vary in abundance and intracellular localization
•Differing conditions for purifying individual proteins.
Two major considerations relating to protein-protein complexes
•Have to identify protein-protein interactions
•Characterize the molecular and biophysical interactions between proteins
Molecular and biophysical characterization of complexes:
additional considerations
•Oligomeric state of interacting proteins
•Stoichiometry of the complex
•Affinity of interacting partners for each other
•In vitro analyses require large amounts of pure proteins
The two general themes of interest
•Methods for identification of novel protein-protein interactions (“molecular biology” – we’ll touch on this)
•Methods for analyses of protein-protein interactions (“cell biology” and “physical biochemistry” – major focus of the lecture)
Methods for identification of novel protein-protein interactions
Identifying novel protein-protein interactions: tandem affinity
purification
Large-scale identification of protein-protein complexes
•Gavin et al. (Nature 415: 141-147, 2002) performed massive TAP strategy using yeast S. cerevisiae
•Tag one component of 200 different complexes, transformation, perform TAP, identify subunits by mass spectrometry
Strategy of Gavin et al.
Gavin et al., Nature 415:141-147
A partial interactome of S. cerevisiae
Methods for analyses of protein-protein interactions: in vitro
approaches
Surface plasmon resonance (SPR)
http://www.astbury.leeds.ac.uk/Facil/SPR/spr_intro2004.htm
• SPR occurs when light is reflected off thin metal film
• Fraction of light energy interacts with delocalized electrons in metal and angle at which this occurs is determined by refractive index on backside of film
• Molecule binding to surface changes refractive index, leading to change in .
• Monitored in real time as changes in reflected light intensity to produce a sensorgram
Advantages of SPR
•No labeling of interacting proteins required (can even use cell extracts)
•Interactions detected in real time
•Both equilibrium and interaction kinetics can be analyzed
•But.....one protein must be tethered to the surface
Example of SPR: SDF-1 binding to chemokine receptor CXCR4
Stenlund et al. Anal. Biochem. 316: 243-250, 2003
Stenlund et al. Anal. Biochem. 316: 243-250, 2003
Lipid bilayer?
yes
no
no
Stenlund et al. Anal. Biochem. 316: 243-250, 2003
Methods for analyses of protein-protein interactions: in vivo
approaches
Fluorescencecorrelationspectroscopy(FCS)
G(), is a measure of the self-similarity ofthe signal after a lag time (). It resemblesthe conditional probability of finding a molecule in the focal volume at a later time.
Dual color cross-correlation: More effective for protein-protein interaction studies
• Cross-correlation amplitude is proportional to number of double-labeled molecules
• Suited to monitoring association and dissociation reactions
An example application of FCCS
•Endocytic pathway: cholera toxin (CTX)
•CTX has AB5 subunit structure
•“B” subunit required for membrane binding and cellular uptake
•“A” subunit has enzymatic activity (elevates cAMP) → massive efflux of Na+ and water
•Do subunits remain associated throughout vesicular transport?
Flow chart of the experiment
•Label A and B subunits with indocarbo-cyanine dyes Cy3 and Cy5.
•Double-labeled CTX added to cells
•Wash away excess toxin
•Perform FCCS at successive time points and different positions in same cell
•At what stage in endocytic pathway do “A” and “B” subunits diverge?
Bacia et al. Biophys. J. 83: 1184-1193, 2002
FCCS analyses during the first minute
Bacia et al. Biophys. J. 83: 1184-1193, 2002
FCCS analyses after 15 minutes
Conclusions: endocytosis of CTX, codiffusion in endocytic vesicles,and separation of “A” and “B” subunits within the Golgi apparatus
Note: FRET was not suitable here, since the molecules are not necessarilywithin the “close proximity” required for FRET.
Bacia et al. Biophys. J. 83: 1184-1193, 2002
And at yet later times…..
A brief review of photophysics: Jablonski Diagram
• Photoexcitation from the ground state S0 creates excited states S1, (S2, …, Sn)
• Kasha’s rule: Rapid relaxation from excited electronic and vibrational states precedes nearly all fluorescence emission
• Internal Conversion: Molecules rapidly (10-14 to 10-11 s) relax to the lowest vibrational level of S1.
• Intersystem crossing: Molecules in S1 state can also convert to first triplet state T1; emission from T1 is termed phosphorescence, shifting to longer wavelengths (lower energy) than fluorescence. Transition from S1 to T1 is called intersystem crossing.
What is FRET?
•Fluorescence Resonance Energy Transfer
•Initial energy absoption (excitation)
•Loss of some energy (vibration, etc.)
•Nonradiative movement of energy to second molecule (resonance transfer)
•Loss of some more energy (vibration, etc.)
•Bolus release of remaining energy (emission)
Initial Energy Absorption
donor
donor +h
Single-photonFluorescenceExcitation
h
donor + h
Vibrationalloss
Loss of Some Energy
donor +h
represents the fraction of energythat is not rapidly lost, and isequivalent to the quantum yield
Transfer of Energy to Second Molecule
donor + h
donor
FluorescenceEmission
h
Transfer Efficiency:how much energy is sent to a second moleculeinstead of retained and emitted
donor
acceptor + h
acceptor
h
Loss of Some More Energy
FluorescenceEmission
h
acceptor + h
Release of Remaining Energy
Vibrationalloss
acceptor + h
acceptor + h
acceptor
FluorescenceQuenching
CompleteDissipation
acceptor + h
acceptor represents the quantum yield ofthe acceptor
Energy Distributions During FRET
donor +ET
donor
DonorFluorescenceEmission(FDA)
EDA
donor
acceptor +EDA’
acceptor
EDA’
Vibrationalloss
acceptor + EDA’
EDA’ = EAD
acceptor
Vibrationalloss
AcceptorFluorescenceEmission(FAD)
Directly determined:FDA = donor fluorescence in presence of acceptorFAD = acceptor fluorescence in presence of donor = quantum fluorescent yield of acceptor
Calculated: EDA FDA = energy released by donor EAD FAD = energy released by acceptor
EDA’ = EAD/ = donor energy absorbed by acceptor ET = EDA’+EDA = total energy released by donor
DADA
DAFRET
EE
EE
+=
'
'
DAAD
ADFRET
FF
FE
+=
φφ
//
A cartoon explanation of FRET
• Example: Two membrane-associated proteins. Do they form protein-protein contacts?
• One fused to CFP, the other to GFP
• FRET efficiency is a function of scalar distance apart
FRET: Single-molecule imaging of Ras activation in living cells
Murakoshi et al. PNAS 101: 7317-7322, 2004
Murakoshi et al. PNAS 101: 7317-7322, 2004
Total internal reflection fluorescence
Mashanov et al. Methods 29: 142-152, 2003
• TIRF occurs when light traveling from high- to low-refractive index medium strikes interface at an angle > c (e.g., ~65° for glass-cytoplasm interface)
• High-numerical-aperture lens → light at periphery approaches specimen at i > c causing total internal reflection
• Permits single-molecule imaging of cell surface phenomena with unparalleled resolution
high low
An example application of TIRFM
•Early events in signal transduction
•Binding of epidermal growth factor (EGF) to its receptor (EGFR)
•EGF binds EGFR:EGF-(EGFR)2 + EGF → (EGF-EGFR)2
or2(EGF-EGFR) → (EGF-EGFR)2 ??
•Autophosphorylation of EGFR →→→ cell division
•Examined Cy3-EGF binding to EGFR by TIRFM
The experimental set up of Sako et al.
Sako et al. Nature Cell Biol. 2: 168-172, 2000
Sako et al. Nature Cell Biol. 2: 168-172, 2000
Their data favor the followingbinding mechanism:
EGF-(EGFR)2 + EGF → (EGF-EGFR)2