A constant-time three-dimensional triple-resonance pulse scheme to correlate intraresidue 1H N, 15N, and 13C′ chemical shifts in 15N 13C-labelled proteins

Abstract

Listen

Translate article

Sequence-specif ic resonance assignments are a prerequisite for structural and dynamical interpretation of protein NMR spectra. For proteins smaller than 10 kDa assignment strategies have relied upon through-bond correlations in homonuclear COSY and TOCSY spectra to identify resonances associated with particular spin systems. Conformation-dependent nuclear Overhauser effects are then emp loyed to sequentially connect these spin systems (1-5). In larger proteins, however, extensive resonance overlap and decreased sensitivity of experiments utilizing ‘H‘H scalar couplings have hindered this approach. ‘HI%15N triple-resonance experiments provide a conformation-independent approach for the assignment of backbone resonances in I%15N-labeled large proteins (6-13). In addition these experiments allow accurate measurement of coupling constants in proteins with large linewidths (14, 15). These experiments exploit large heteronuclear one-bond couplings to transfer magnetization with the sensitivity of indirect detection. As demonstrated in calmodulin ( 16, 17) backbone assignments utilize four tr iple-resonance experiments [ HNCa, HNCO, HCaCO, and HCA(CO)N] and the 3D TOCSY-HMQC experiment ( 18). A fifth tr iple-resonance experiment [ H( CA)NHN] is a useful complement to the TOCSY-HMQC experiment, correlating ‘HN “N and ‘H” resonances ( 7, 9, 13). Furthermore, a sixth experiment, the HN (CO ) CA. has been introduced, providing additional sequential information ( 11) . Of the experiments listed above, those which detect am ide protons [ HNCA, HNCO, H(CA)NHN, HN(CO)Ca] need to be collected in H20, while those with detection of (Y protons [ HCACO and HCA( CO)N] are best performed in 2H20. This causes difficulties if assignments are based on al ignment of C’ chemical shifts, because these resonances may show significant isotope shifts, depending on whether the carbonyl is hydrogen bonded to a proton or a deuteron. It is therefore desirable to perform the ma jority of experiments in the same solvent ( H20), restricting oneself to those experiments which detect am ide protons [ HNCA, HNCO, HN( CO)CA. H( CA )NHN 1.