Measurement of signs of chemical shift differences between ground and excited protein states: a comparison between H(S/M)QC and R1rho methods.
Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR spectroscopy has emerged as a powerful tool for quantifying the kinetics and thermodynamics of millisecond exchange processes between a major, populated ground state and one or more minor, low populated and often invisible 'excited' conformers. Analysis of CPMG data-sets also provides the magnitudes of the chemical shift difference(s) between exchanging states (|Deltavarpi|), that inform on the structural properties of the excited state(s). The sign of Deltavarpi is, however, not available from CPMG data. Here we present one-dimensional NMR experiments for measuring the signs of (1)H(N) and (13)C(alpha) Deltavarpi values using weak off-resonance R (1rho ) relaxation measurements, extending the spin-lock approach beyond previous applications focusing on the signs of (15)N and (1)H(alpha) shift differences. The accuracy of the method is established by using an exchanging system where the invisible, excited state can be converted to the visible, ground state by altering conditions so that the signs of Deltavarpi values obtained from the spin-lock approach can be validated with those measured directly. Further, the spin-lock experiments are compared with the established H(S/M)QC approach for measuring the signs of chemical shift differences. For the Abp1p and Fyn SH3 domains considered here it is found that while H(S/M)QC measurements provide signs for more residues than the spin-lock data, the two different methodologies are complementary, so that combining both approaches frequently produces signs for more residues than when the H(S/M)QC method is used alone.
Auer
Renate
R
Department of Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, Campus-Vienna-Biocenter 5, 1030, Vienna, Austria.
Hansen
D Flemming
DF
Neudecker
Philipp
P
Korzhnev
Dmitry M
DM
Muhandiram
D Ranjith
DR
Konrat
Robert
R
Kay
Lewis E
LE
D015233:Models, Statistical; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D013816:Thermodynamics
D009587:Nitrogen Isotopes; D011506:Proteins