Conformational Analysis of Uniformly 13C-Labeled Peptides by Rotationally Selected 13Cα-13CH3 Double-Quantum Solid-State NMR

TL;DR

This paper introduces a new solid-state NMR method to determine the 3D structure of labeled peptides using carbon-carbon and carbon-hydrogen interactions.

Contribution

A novel MAS SSNMR approach using rotational resonance and double-quantum coherence for peptide structure determination in the solid state.

Findings

The method measures 13C–13C distances and C–H bond orientations in uniformly 13C-labeled peptides.

Two molecular conformations of fMLF were determined, one matching a related peptide's crystal structure.

The technique is applicable to amyloid fibrils and pharmaceutical formulations.

Abstract

Peptides are an important class of biomolecules that perform many physiological functions and which occupy a significant and increasing share of the pharmaceutical market. Methods to determine the solid-state structures of peptides in different environments are important to help understand their biological functions and to aid the development of drug formulations. Here, a new magic-angle spinning (MAS) solid-state nuclear magnetic resonance (SSNMR) approach is described for the structural analysis of uniformly 13C-labeled solid peptides. Double-quantum (DQ) coherence between selective pairs of 13C nuclei in peptide backbone and side-chain CH3 groups is excited to provide restraints on (i) 13C–13C internuclear distances and (ii) the relative orientations of C–H bonds. DQ coherence is selected by adjusting the MAS frequency to the difference in the resonance frequencies of selected nuclear pairs (the rotational resonance condition), which reintroduces the dipolar coupling between the nuclei. Interatomic distances are then measured using a constant time SSNMR experiment to eliminate uncertainties arising from relaxation effects. Further, the relative orientations of C–H bond vectors are determined using a DQ heteronuclear local field SSNMR experiment, employing 13C–1H coupling amplification to increase sensitivity. These methods are applied to determine the molecular conformation of a uniformly 13C-labeled peptide, N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLF). From just six distance and six angular restraints, two possible molecular conformations are determined, one of which is in excellent agreement with the crystal structure of a closely related peptide. The method is envisaged to a useful addition to the SSNMR repertoire for the solid-state structure determination of peptides in a variety of forms, including amyloid fibrils and pharmaceutical formulations.

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