Side chain dynamics monitored by 13C-13C cross-relaxation

Abstract

A method to measure 13C-13C cross-relaxation rates in a fully 13C labeled protein has been developed that can give information about the mobility of side chains in proteins. The method makes use of the (H)CCH-NOESY pulse sequence and includes a suppression scheme for zero-quantum (ZQ) coherences that allows the extraction of initial rates from NOE buildup curves. The method has been used to measure 13C-13C cross-relaxation rates in the 269-residue serine-protease PB92. We focused on Cα-Cβ cross-relaxation rates, which could be extracted for 64% of all residues, discarding serine residues because of imperfect ZQ suppression, and methyl 13C-13C cross-relaxation rates, which could be extracted for 47% of the methyl containing C-C pairs. The Cα-Cβ cross-relaxation rates are on average larger in secondary structure elements as compared to loop regions, in agreement with the expected higher rigidity in these elements. The cross-relaxation rates for methyl containing C-C pairs show a general decrease of rates further into the side chain, indicating more flexibility with increasing separation from the main chain. In the case of leucine residues also long-range Cβ-Cδ cross-peaks are observed. Surprisingly, for most of the leucines a cross-peak with only one of the methyl Cδ carbons is observed, which correlates well with the χ2 torsion-angle and can be explained by a difference in mobility for the two methyl groups due to an anisotropic side chain motion.