Accurate Measurement of Alpha Proton Chemical Shifts of Excited Protein States by Relaxation Dispersion NMR Spectroscopy
2009-02-11T00:00:00Z (GMT) by
Carr−Purcell−Meiboom−Gill relaxation dispersion NMR spectroscopy can provide detailed information about low populated, invisible states of protein molecules, including backbone chemical shifts of the invisible conformer and bond vector orientations that can be used as structural constraints. Notably, the measurement of 1Hα chemical shifts in excited protein states has not been possible to date because, in the absence of suitable labeling, the homonuclear proton scalar coupling network in side chains of proteins leads to a significant degradation in the performance of proton-based relaxation dispersion experiments. Here we have overcome this problem through a labeling scheme in which proteins are prepared with U−2H glucose and 50% D2O/50% H2O that results in deuteration levels of between 50−88% at the Cβ carbon. Effects from residual 1Hα−1Hβ scalar couplings can be suppressed through a new NMR experiment that is presented here. The utility of the methodology is demonstrated on a ligand binding exchanging system and it is shown that 1Hα chemical shifts extracted from dispersion profiles are, on average, accurate to 0.03 ppm, an order of magnitude better than they can be predicted from structure using a database approach. The ability to measure 1Hα chemical shifts of invisible conformers is particularly important because such shifts are sensitive to both secondary and tertiary structure. Thus, the methodology presented is a valuable addition to a growing list of experiments for characterizing excited protein states that are difficult to study using the traditional techniques of structural biology.