J. Am. Chem. Soc., 2009, 131 (31), pp 10832–10833

Measuring the Signs of 1Hα Chemical Shift Differences Between Ground and Excited Protein States by Off-Resonance Spin-Lock R1ρ NMR Spectroscopy

Renate Auer†‡, Philipp Neudecker‡, D. Ranjith Muhandiram‡, Patrik Lundstrm‡§, D. Flemming Hansen‡, Robert Konrat† and Lewis E. Kay*‡

Analysis of Carr−Purcell−Meiboom−Gill (CPMG) relaxation dispersion NMR profiles provides the kinetics and thermodynamics of millisecond-time-scale exchange processes involving the interconversion of populated ground and invisible excited states. In addition, the absolute values of chemical shift differences between NMR probes in the exchanging states, |Δ|, are also extracted. Herein, we present a simple experiment for obtaining the sign of 1Hα Δ values by measuring off-resonance 1Hα decay rates, R1ρ, using weak proton spin-lock fields. A pair of R1ρ values is measured with a spin-lock field applied |Δω| downfield and upfield of the major-state peak. In many cases, these two relaxation rates differ substantially, with the larger one corresponding to the case where the spin-lock field coincides with the resonance frequency of the probe in the minor state. The utility of the methodology is demonstrated first on a system involving protein ligand exchange and subsequently on an SH3 domain exchanging between a folded state and its on-pathway folding intermediate. With this experiment, it thus becomes possible to determine 1Hα chemical shifts of the invisible excited state, which can be used as powerful restraints in defining the structural properties of these elusive conformers.