multidomain proteins and solution methods for studying protein structure

what does proteins being multivalent mean

can bind another biomolecule through multiple sites

what is avidity

total strength of binding affinity

what is c300/CBP

a large multidomain modular protein with a KIX domain

KIX domain

well-studied by solution-based approaches

example o f a small domain that can bind to different peptide motifs via 2 different binding surfaces

interaction between KIX and pKID (domain of CREB)

occurs via a hydrophobic surface

mechanism is uncertain (fold before binding or fold on surface of KIX)

pKID

unstructured

phosphorylation of Ser133 increases affinity 100 fold

Kd = 0.7um

DSF

measures fluorescence of trp side chain groups at 2 wavelengths, as they fluoresce differently whether they are in a hydrophobic or polar environment

how can melting temp (TM) of a protein be determined

by heating a sample and following the ratio of fluorescence emission at 330 and 350 nm

circular dichroism

exploits the fact that different protein secondary structures absorb circularly polarised light in a characteristic way

gives the percentage of each secondary structure type in a protein and can be used to monitor changes in secondary structure

small angle x-ray scattering SAXS

solution-based scattering technique that provides low resolution structural info about a protein or protein complex

also to monitor changes in global structure that occur due to a ligand or partner binding, PTM or mutation