using chemical shift to learn about protein structure and function

what can be measured using NMR spec

structure, interaction, dynamics, it measures nuclear spin

sample requirements for NMR spec experiments

isotopically enrich proteins with 13C and 15N

produce recombinant samples with bacterial, yeast or eukaryotic hosts

>200uL of protein

low ionic strength, non organic and low pH buffer

>10% D2O

flow chart of NMR project

sample prep

1/2/3D NMR spec

resonance assignment

determine interactions, 3D structure, dynamics

how can biomolecular interactions be studied using NMR spec

monitor chemical shift changes between free and bound states

how can the binding interface be shown

if backbone resonance assignments are available, chemical shift changes can be mapped on a 3D structure of on of the binding partners

how can a binding affinity be calculated

NMR titration, plot binding isotherm from chemical shift changes

what does a non-linear chemical shift change in NMR titrations suggest

coupled folding and binding