Protein-Protein Interactions Assay

Why PPI (Protein Protein Interaction)?

· Protein-Protein Interactions determine the outcome of most cellular processes
· Proteins which are close homologues often interact in the same way
· Protein-Protein Interactions place evolutionary constraints on protein sequence and structural divergence
· Pre-cursor to networks

PPI (Protein Protein Interaction) Classification

· Strength of interaction — Permanent or transient
· Specificity
· Location within polypeptide chain
· Similarity of partners – Homo- or hetero-oligomers
· Direct (binary) or a complex
· Confidence score

Protein-Protein Interactions Assay

Determining PPIs
· Small-Scale Methods

– Co-immunoprecipitation
– Affinity chromatography
– Pull-down assays
– In vitro binding assays – FRET, Biacore, AFM
– Structural (Co-Crystals)

PPIs by High-Throughput Methods

· Yeast two hybrid systems
· Affinity tag purification followed by mass spectrometry
· Protein microarrays
· Microarrays/gene co-expression

– Implied functional PPIs

 · Synthetic lethality

– Genetic interactions, implied functional PPIs

PPI (Protein Protein Interaction) Lab Experiments

• Small-scale PPI experiments
• One protein or several proteins at a time
• Small amount of available data
• Expensive and slow lab process
• High-throughput PPI experiments
• Hundreds/thousands of proteins at a time
• Highly noisy and incomplete data
• Surprisingly little overlap among different sets

Yeast Two Hybrid System

Yeast transcription factor has a binding domain (BD) and activation domain (AD)

– BD binds to upstream of the target gene on DNA
– AD is required to activate transcription
– BD and AD function independently

Advantages

– Fairly simple, rapid and inexpensive
– Requires no protein purification
– No previous knowledge of proteins needed
– Scalable to high-throughput
– Is not limited to yeast proteins

Limitations

– Works best with cytosolic proteins
– Tendency to produce false positives

Mass Spectrometry

· Need to purify protein or protein complexes
· Use a affinity-tag system
· Need efficient method of recovering fusion protein in low concentration

Mass Spectrometry (MS) of Purified Complexes

• Affinity Purification and Mass Spectrometry

– Multiprotein complexes are isolated directly from cell lysates through one or more affinity purification steps
– Complex components are then identified by MS

• Unlike Two-Hybrid Assay,

– MS can be performed under near physiological conditions in the relevant organism and cell type
– MS does not perturb post translational modification, thus the effects of post translational modification can be detected

TAP

Advantages

– No prior knowledge of complex composition
– Two-step purification increases specificity of pull-down

Limitations

– Transient interactions may not survive 2 rounds of washing
– Tag may prevent interactions
– Tag may affect expression levels
– Works less efficiently in mammalian cells

Other tags

HA, Flag and His

– Anti-tag antibodies can interfere with MS analysis

Streptavidin Binding Peptide (SBP)

– High affinity for streptavidin beads
– 10-fold increase in efficiency of purification compared to conventional TAP tag
– Successfully used to identify components of complexes in the Wnt/b-catenin pathway

Databases of Protein-Protein Interactions

MINT – Molecular Interaction Database

->240,000 interactions with 35,000 proteins

– Covers multiple species

DIP — Database of Interacting Proteins (UCLA)

->79,000 interactions with >27,000 proteins

CCSB – Proteomics Base Interactomes (Harvard)

– Human, viruses, C. elegans, S. cerevisiae
– Some unpublished data

IntAct – EBI molecular interaction database
-Curated data from multiple sources