Determination of Leu Side-Chain Conformations in Excited Protein States by NMR Relaxation Dispersion

摘要

Protein function is often predicated on the interconversionnbetween highly populated, ground state conformers and lowpopulated,nexcited state structures. These excited states are ‘invisible’nto most of the traditional tools of structural biology but theyncan be characterized by the Carr-Purcell-Meiboom-Gill (CPMG)nrelaxation dispersion NMR experiment,1 so long as their lifetimesnare between 0.5 and 10 ms and their population (pE) exceeds 0.5%.nThe kinetics and thermodynamics of a variety of processes involvingnexcited states, including enzyme catalysis,2,3 ligand binding,4,5 andnprotein folding,6 have been derived from CPMG experiments. Thesenexperiments also provide chemical shifts and orientations of bondnvectors of the excited state, which form the basis for structurendetermination of these invisible conformers.7,8 Very recently it hasnbeen shown that for small proteins with pE > 2-3% and exchangenrates on the order of several hundred/s it is possible to extractnmethyl-containing side-chain dynamics parameters of the excitednstate,21 relating directly to the conformational entropy of the sidechain.n9 Herein, we extend the methodology by providing a verynsensitive approach for determining the relative rotamer populationsnof Leu side-chains in low populated, invisible states.