10.23.26 - Methods Studying Protein-Protein Interactions – Axel Mogk

three reasons to study protein-protein interactions

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Proteins function primarily in complexes as opposed to in isolation 

Complexes are dynamic and dependent on growth conditions 

Defining complexes is crucial for understanding cellular function and physiology – creation of interaction network 

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genetic hint

"genetic data used to start study/deduce protein interactions "

what are two major genetic hints for protein-protein interaction?

gene co-regulation or co-organization

why would gene co-regulation be a genetic hint?

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Proteins act in same cellular pathway, could be due to complex formation 

If interacting, gene expression patterns should be similar (or influenced by each other) 

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why would gene co-organization be a genetic hint?

Genes in operons frequently form complexes 

Gene knockouts lead to same phenotypes 

Likely that proteins act in same cellular pathway 

what are some genetic methods of studying protein-protein interactions?

genetic hints, suppressor mutations, synthetic genetic arrays, Y2H and derivatives

suppressor mutations

Mutating one partner protein from WT, mutating extragenic interaction partner (interaction suppressor, allele-specific) to complement mutant and restore function

"how does mutating or overexpressing an extragenic interaction partner restore function?"

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Unstable mutant protein can be stabilized by interaction with partner protein 

Degradation is associated with mutant phenotype 

Increased partner protein expression results in WT phenotype 

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synthetic genetic arrays

"Single knockout of two genes separately result in mild phenotype – double knockout created and phenotype scored "

what are the three results of synthetic genetic arrays?

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same as single knockouts – no genetic interaction 

sickness, lethality, or a worse phenotype (aggravating) – negative genetic interaction – parallel cellular pathways, proteins do not interact 

better phenotype (alleviating) - positive genetic interaction 

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why is a negative genetic interaction a sign that two proteins do NOT interact?

"If interacting, a single knockout and a double knockout would result in same phenotype as both cause total complex inactivation "

what can be done with a set of knockout scores? (SGA)

"Heat map made of knockout scores with one gene knockout combined with many potential others and genetic interaction map can be made "

what gene is used in classical Y2H?

"modular domain organization of Gal4 transcription activator - DBD and AD"

"Yeast-two-hybrid assay"

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1. Separate DNA-binding and activator domains, fuse to proteins - DBD-bait and AD-prey (potential binding partner)
2. Reporter screen – reporter gene is placed under control of promoter that Gal4 interacts with 
3. Bait-DBD binds to promoter upstream activator sequence 
4. IF bait and prey proteins interact, AD triggers gene expression via RNA Pol II 

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what gene is typically used as reporter for Y2H and what are the bacterial growth conditions and indicators?

lacZ - encodes B-galactosidase (which splits lactose into glucose, galactose, and allolactase)
LB agar plates with XGal (substrate), interaction indicated by growth of blue colonies
MacConkey agar plates with 1% Lactose, interaction indicated by growth of red colonies (lactose fermentation)

"how do prototrophy reporters (HIS3, LEU2, URA3, ADE2) work in Y2H?"

if placed in specific-aa deficient mutants and media, growth of cells indicate the ability to express reporter gene due to bait-prey interaction

B2H systems

"use Bordetalla pertussis adenylyl cyclase

Adenylyl cyclase catalytic domain can be separated into inactive T18 and T25 

Activity restored via protein interaction 

cAMP binds to and activates CAP TF of reporter gene

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causes of 2H screen false positives

Often overexpression of bait and preys 

Interaction forced in nucleus, wrong physiological compartment 

Unspecific interactions, recurrent false positives 

causes of 2H screen false negatives

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Interaction takes place outside nucleus 

Different cellular environment needed to non-yeast/bacterial proteins (PTMs) 

Interaction only occurs in context of larger protein complex 

Tagging of proteins prevents interactions 

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Classical Co-IP

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1. Cell lysate 
2. Incubate lysate with bait-specific antibody
3. Remove unbound proteins, isolate antibody complex through centrifugation and washing
4. Western blot against prey protein 
5. Mass spec analysis 

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"Antibody generation methods "

"Inject purified protein (native or denatured) into animal (rabbit, guinea pig) along with substances that stimulate immune response 
Inject protein-derived peptides linked to inert carrier into animal 
N- or C- terminal fusion of affinity tags (Myc, HA, FLAG) to protein of interest "

how are antibodies coupled to agarose beads?

"Carbonyl on beads react covalently with anime (lysine) on antibody 
Bacterial protein A/G is covalently coupled to beads, constant region of antibody (Fc on heavy chain) binds to protein "

where do proteins A and G orginate from?

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Protein A – staphylococcus aureus 

Protein G – streptococcus spp. 

Surface proteins, important for pathogenesis, phagocytosis reduction, defending against host immune response 

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Avi-tag

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Lysine is biotinylated by BirA biotin ligase 

Can be isolated by binding to streptavidin or avidin 

Very high affinity – 10 ^-15 M Kd 

Purification can occur even under denaturing conditions 

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"Glutathione-S-Transferase pulldown"

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1. GST-bait fusion expressed in E.coli 
2. wash with glutathione particles and mix with glutathione-sepharose beads (isolation)
3. Incubate with prey lysate 
4. Prey obtained using plasmid 
5. Wash, elute, western 

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pros of tandem-affinity purification

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Specificity increased and background reduced by two consecutive purification steps 

Mild elution conditions, maintains complex integrity 

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historical TAP-tag structure

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Calmodulin-binding peptide - Binds CaM in Ca dependent manner 

Tobacco Etch Virus protease - Binds consensus sequence 

ProtA fragment 

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TAP-tag purification protocol

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1. CBP bound to bait 
2. ProtA binds antibody beads 
3. TEV protease cleavage to release beads 
4. Bind to CaM beads w/Ca 
5. Chelate with EGTA 

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historical TAP-tag concerns

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Large size 

EGTA might affect cation dependent proteins 

Many proteins bind to CaM  

Low recovery in mammalian cells (1%) 

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how to use TAP-tag under denaturing conditions

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Crosslink in vivo with formaldehyde 

Sometimes protein-protein interactions are too transient 

His and biotinlyation tags 

8M Urea conditions 

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"biochemical methods of protein-protein interaction detection "

classical Co-IP, protein based pulldown methods, GST pulldowns, tandem-affinity purification

cell biological methods of determining protein-protein interactions

in vivo FRET, protein fragment complementation assays, proximity labeling, proximity ligation

in vivo FRET

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Excitation of donor fluorophore which transmits energy to acceptor fluorophore 

Donor bound to bait, acceptor bound to prey 

Typically CFP/YFP or GFP/RFP 

Low nm range required 

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"Protein fragment complementation assay"

"Fluorescent sensor split into fragments and bound to bait and prey "

"Protein fragment complementation assay advantages and disadvantages"

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Advantage - Shows subcellular localization 

Disadvantage - Limited by steric hinderance and activation is irreversible (false positives) 

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"Protein fragment complementation assay proteins/examples"

"GFP, luciferase, Ub, DHFR, Venus"

proximity labeling

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Fuse bait with enzyme that creates small reactive labels 

Isolate and identify with mass spec 

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reaction for BioID and APEX proximity labeling techniques

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BioID - BirA* biotinlyating enzyme fusion protein (engineered to have relaxed selectivity from Avi tag) 

In presence of biotin, 10 nm radius of reactive biotinyl-5-AMP is created  

APEX - Ascorbate peroxidase 

Oxidizes biotin-phenol to reactive phenol radicals 

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proximity ligation

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1. Specific antibodies against bait and prey 
2. Secondary antibody conjugated with oligonucleotide 
3. Add connector oligonucleotides 
4. Amplify new formed circular DNA molecule 
5. Detection by fluorescently labeled complementary oligonucleotides 

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"Proximity ligation characteristics"

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Distance up to 30nm 

Highly sensitive 

Does not allow to distinguish interaction from proximity 

Can control with only primary, only secondary, or with knockout 

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Site-specific labeling of cysteins with UV-activatable crosslinker

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1. Introduce cysteine at positions of interest 
2. Label protein variants with UV-activatable crosslinker 
3. Separate labeled protein from free label 
4. Perform crosslinking experiment 
5. Analyze w/ western blot, mass spec 

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Disuccinimidyl suberate

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Homobifunctional crosslinker for primary amines 

Works up to 30 A 

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DSS crosslinking protocol

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1. Crosslink proteins in target complex 
2. Trypsin digest 
3. LC-MS/MS 
4. Database searching 

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main pro of crosslinking peptides

"Captures highly transient interactions "

peptide libraries

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Represent primary sequence as overlapping peptides covalently coupled to membrane 

Incubation of library to proteins of interest along w/ western blots shows which peptides interact 

interaction can be double checked by mutation

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