Abstract
Resonance assignment and structural studies of larger proteins by nuclear magnetic resonance (NMR) can be challenging when exchange broadening, multiple stable conformations, and H back-exchange of the fully deuterated chain pose problems. These difficulties arise for the SARS-CoV-2 Main Protease, a homodimer of 2 306 residues. We demonstrate that the combination of four-dimensional (4D) TROSY-NOESY-TROSY spectroscopy and 4D NOESY-NOESY-TROSY spectroscopy provides an effective tool for delineating the H-H dipolar relaxation network. In combination with detailed structural information obtained from prior X-ray crystallography work, such data are particularly useful for extending and validating resonance assignments as well as for probing structural features.
Highlights
The extension of conventional two-dimensional 1H–1H nuclear magnetic resonance (NMR) spectroscopy of natural proteins (Wüthrich, 1986) to three-dimensional (3D) homonuclear NMR experiments offered the ability to simplify spectral analysis by removing resonance overlap (Vuister et al, 1988; Oschkinat et al, 1988) and by providing access to a direct, more detailed analysis of 1H–1H dipolar cross-relaxation networks
We demonstrate the utility of the experiments by applying them to the study of the main protease of SARS-CoV2 (Mpro), which is the virus responsible for coronavirus2019 disease (COVID-19)
The fusion protein encoded for 6His tag – GB1–SG rich linker–TEV cleavage site – MpCr1o45A and was purified according to methods collectively developed by the COVID-19 NMR consortium (Altincekic, 2021)
Summary
The extension of conventional two-dimensional 1H–1H NMR spectroscopy of natural proteins (Wüthrich, 1986) to three-dimensional (3D) homonuclear NMR experiments offered the ability to simplify spectral analysis by removing resonance overlap (Vuister et al, 1988; Oschkinat et al, 1988) and by providing access to a direct, more detailed analysis of 1H–1H dipolar cross-relaxation networks. This information complemented and validated the elegant relaxation matrix analysis of spin diffusion (Boelens et al, 1988) Such homonuclear 1H 3D experiments and analysis strategies were soon followed by a myriad of heteronuclear 3D experiments that required isotopic enrichment and cloning and bacterial overexpression (Marion et al, 1989b; Zuiderweg and Fesik, 1989; Ikura et al, 1990; Marion et al, 1989a; Wagner, 1993). Most of these heteronuclear experiments served to disperse the regular 1H–1H 2D spectrum into a third dimension, thereby removing spectral overlap but providing little or no new information on the all-important 1H–1H spin-diffusion pathways. The 3D NOESY-HMQC experiment (Marion et al, 1989b; Zuiderweg and Fesik, 1989) subsequently was extended to four dimensions (4D), thereby dispersing the conventional 2D 1H– 1H NOESY experiment into two additional dimensions that correspond to the chemical shifts of the nuclei to which each of the protons is covalently bound (Kay et al, 1990; Clore et al, 1991; Zuiderweg et al, 1991)
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