W10 Methods to study Protein Protein Interactions in Vitro Flashcards
characteristics of protein-protein interactions
binding surface is exposed before/upon binding
binding surfaces has “hot spots”
binding induced conformational changes
binding affinity is highly diverse: very weak to very strong
bindings/interactions can be non-covalent and reversible
types of protein-protein interactions
hetero-oligomers: interactions of different protein subunits
homo-oligomers: interactions of identical protein subunits
transient
stable
covalent
non covalent
what is isothermal titration calorimetry (ITC)
method to directly measure heat change during binding/ interaction between 2 molecules
used for protein-small molecules, protein-protein, antibody-antigen, protein-dan, protein-lipids
no labelling or chemical modification
easy handling: unattended operation after sample loading
types of peaks in ITC profile
exothermic peaks: peaks are downward
endothermic peaks: peaks are upwards
conditions for sample preparation in ITC
samples are prepared in standard buffers (phosphate buffers, tris-buffers etc)
sample concentrations: micro-molar range
concentration of interacting molecule in syringe should be 10 times higher than that of sample in cell
small molecules (drugs or short peptides) placed in syringe while larger molecules (proteins, dna) kept in cell
all samples should be made in same buffer with same pH (mixing different buffer may yield additional heat)
limitations of ITC to determine binding affinity
unable to determine binding parameters for low affinity complexes due to lack of saturation
unable to determine binding parameters for very high affinity complex due to saturation being too quickly
what is microscale thermophoresis
method used to study interactions by measuring movement of molecules in response to temperature gradient
when molecule interacts with another molecule > effective size, charge or shape may change > influence how molecule responds to the temperature gradient
