Measuring the signs of 1H(alpha) chemical shift differences between ground and excited protein states by off-resonance spin-lock R(1rho) NMR spectroscopy.
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, |Delta omega|, are also extracted. Herein, we present a simple experiment for obtaining the sign of (1)H(alpha) Delta omega values by measuring off-resonance (1)H(alpha) decay rates, R(1rho), using weak proton spin-lock fields. A pair of R(1rho) values is measured with a spin-lock field applied |Delta omega| 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 (1)H(alpha) chemical shifts of the invisible excited state, which can be used as powerful restraints in defining the structural properties of these elusive conformers.
Auer
Renate
R
Department of Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, Campus-Vienna-Biocenter 5, A-1030 Vienna, Austria.
Neudecker
Philipp
P
Muhandiram
D Ranjith
DR
Lundström
Patrik
P
Hansen
D Flemming
DF
Konrat
Robert
R
Kay
Lewis E
LE
D007700:Kinetics; D008722:Methods; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D013816:Thermodynamics
D011506:Proteins