Methods to Study Protein-Protein Interactions Dr. Michal Štros Biofyzikální ústav AV ČR, Brno Protein-Protein Interactions: The “Interactome” 2 challenges: - find which proteins interact (the partners) - find which residues participate in the interactions protprot Studying protein-protein interactions • • 1. 2-hybrid system • 2. Antibody-array • 3. Pull-down • 4. Immunoprecipitation • 5. FRET • • • DNA Binding Domain Activation Domain Yeast 2-hybrid system DNA Binding Domain Activation Domain DNA Binding Domain Prot1 Activation Domain Prot2 Yeast 2-hybrid system Binding Domain Activation Domain Binding Domain Prot1 Activation Domain Prot2 Reporter Gene Promoter Region DNA Binding Domain Prot1 Activation Domain Prot2 mRNA Yeast 2-hybrid system If Prot1 and Prot2 interact: Yeast 2-hybrid system • A transcription factor is split into 2 domains • • 2 hybrid proteins are designed, each containing one of the two proteins that are tested • • If the two proteins interact, the two domains from the transcription factor will interact, causing expression of a (detectable) reporter gene • • The reporter can be: - essential, in which case the yeast colony dies if the 2 proteins do not interact - reversely, the reporter gene can be attached to a green fluorescent protein Unfortunately, the rate of false positive is high (estimated > 45%) DNA Binding Domain Activation Domain Mammalian 2-hybrid system Antibody-Array Interaction Mapping Pull-down assay in vitro or Immunoprecipitation of proteins expressed in the cell FRET(Fluorescence Resonance Energy Transfer) FRET(Fluorescence Resonance Energy Transfer) When will FRET occur? 1) Spectral overlap Donor emission spectrum must significantly overlap the absorption spectrum of the acceptor (>30%) 2) Distance between the donor and acceptor is between 2 - 10 nm 3) Favorable orientation of fluorophores 2 ~ 10 nm Donor emission Acceptor absorption Feasible chromophore-pairs in FRET studies FLIM (Fluorescence Lifetime Imaging Microscopy)- FRET FRET and Flow-Cytometry Bioluminiscence Resonance Energy Transfer (BRET) BRET for protein-protein interactions imaging in deep-tissues in living mice Bioluminescence images of HT1080 cells stably expressing BRET fusion proteins accumulated in the lungs of nude mice (tail vein injection). Injection of luciferase substrate at 1.5 h after cell injection ( imaged using sequentially open/donor/akceptor filters) BRET for protein-protein interactions imaging in deep-tissues in living mice Bioluminescence images of HT1080 cells stably expressing BRET fusion proteins accumulated in the lungs of nude mice (tail vein injection). Injection of luciferase substrate at 1.5 h after cell injection ( imaged using sequentially open/donor/akceptor filters) FRAP (Fluorescence Recovery After Photobleaching)