Measurement of the signs of methyl 13C chemical shift differences between interconverting ground and excited protein states by R1ρ: an application to αB-crystallin

Carr-Purcell-Meiboom-Gill relaxation dispersion (CPMG RD) NMR spectroscopy has emerged as a powerful tool for quantifying the kinetics and thermodynamics of millisecond time-scale exchange processes involving the interconversion between a visible ground state and one or more minor, sparsely populated invisible 'excited' conformational states. Recently it has also become possible to determine atomic resolution structural models of excited states using a wide array of CPMG RD approaches. Analysis of CPMG RD datasets provides the magnitudes of the chemical shift differences between the ground and excited states, Delta, but not the sign. In order to obtain detailed structural insights from, for example, excited state chemical shifts and residual dipolar coupling measurements, these signs are required. Here we present an NMR experiment for obtaining signs of C-13 chemical shift differences of (CH3)-C-13 methyl groups using weak field off-resonance (1) relaxation measurements. 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 sample conditions so that the signs of Delta values obtained from the spin-lock approach can be validated against those measured directly. Further, the spin-lock experiments are compared with the established H(S/M)QC approach for measuring signs of chemical shift differences and the relative strengths of each method are discussed. In the case of the 650 kDa human alpha B-crystallin complex where there are large transverse relaxation differences between ground and excited state spins the (1) method is shown to be superior to more 'traditional' experiments for sign determination.