Intra-residue methyl\u2013methyl correlations for valine and leucine residues in large proteins from a 3D-HMBC-HMQC experiment

Lucas Siemons,Vaibhav Kumar Shukla,D Flemming Hansen,Harold W Mackenzie

doi:10.1007/s10858-019-00287-9

Lucas Siemons, Vaibhav Kumar Shukla + Show 2 more

Open Access

https://doi.org/10.1007/s10858-019-00287-9

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Abstract

Methyl-TROSY based NMR experiments have over the last two decades become one of the most important means to characterise dynamics and functional mechanisms of large proteins and macromolecular machines in solution. The chemical shift assignment of methyl groups in large proteins is, however, still not trivial and it is typically performed using backbone-dependent experiments in a ‘divide and conquer’ approach, mutations, structure-based assignments or a combination of these. Structure-based assignment of methyl groups is an emerging strategy, which reduces the time and cost required as well as providing a method that is independent of a backbone assignment. One crucial step in available structure-based assignment protocols is linking the two prochiral methyl groups of leucine and valine residues. This has previously been achieved by recording NOESY spectra with short mixing times or by comparing NOESY spectra. Herein, we present a method based on through-bond scalar coupling transfers, a 3D-HMBC-HMQC experiment, to link the intra-residue methyl groups of leucine and valine. It is shown that the HMBC-HMQC method has several advantages over solely using NOESY spectra since a unique intra-residue cross-peak is observed. Moreover, overlap in the methyl-TROSY HMQC spectrum can easily be identified with the HMBC-HMQC experiment, thereby removing possible ambiguities in the assignment.

Highlights

The methyl-bearing residues leucine, isoleucine, and valine are often well dispersed throughout a protein and these residues provide significant coverage of the structure
Scalar coupling constants were obtained from Density Functional Theory (DFT) calculations on the Ac-Val-NMe molecule shown in Fig. 1a using the programme Gaussian 09 (Frisch et al 2016)
Density functional theory (DFT) calculations were carried out on the Ac-Val-NMe molecule, Fig. 1a, in order to assess the possibility of obtaining intra-residue through-bond methyl–methyl correlations in large proteins

Summary

Introduction

The methyl-bearing residues leucine, isoleucine, and valine are often well dispersed throughout a protein and these residues provide significant coverage of the structure. Using 13CH3 labelled methyl groups as probes within a uniformly deuterated protein has become an indispensable method to characterise large dynamic systems by Nuclear. The three-fold rotational axis in combination with sophisticated pulse sequences, which maintain the sensitivity enhancement afforded by the methyl-TROSY effect, have provided a suite of experiments that allow one to probe the structure and dynamics in a wide variety of systems (Tugarinov et al 2003; Tugarinov and Kay 2003, 2004; Ruschak and Kay 2010; Hansen and Kay 2011; Hansen et al 2010)

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