Four-dimensional NOE-NOE Spectroscopy of SARS-CoV-2 Main Protease to Facilitate Resonance Assignment and Structural Analysis

Overview

Journal Magn Reson (Gott)

Date 2023 Oct 31

PMID 37904772

Authors

Angus J Robertson

Jinfa Ying

Ad Bax

Affiliations

Soon will be listed here.

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.

Citing Articles

Concordance of X-ray and AlphaFold2 Models of SARS-CoV-2 Main Protease with Residual Dipolar Couplings Measured in Solution.

Robertson A, Courtney J, Shen Y, Ying J, Bax A J Am Chem Soc. 2021; 143(46):19306-19310.

PMID: 34757725 PMC: 8592127. DOI: 10.1021/jacs.1c10588.

References

Barnes C, Shen Y, Ying J, Takagi Y, Torchia D, Sellers J . Remarkable Rigidity of the Single α-Helical Domain of Myosin-VI As Revealed by NMR Spectroscopy. J Am Chem Soc. 2019; 141(22):9004-9017. PMC: 6556874. DOI: 10.1021/jacs.9b03116. View

Lee W, Tonelli M, Markley J . NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy. Bioinformatics. 2014; 31(8):1325-7. PMC: 4393527. DOI: 10.1093/bioinformatics/btu830. View

Pervushin K, Wider G, Wuthrich K . Single Transition-to-single Transition Polarization Transfer (ST2-PT) in [15N,1H]-TROSY. J Biomol NMR. 2010; 12(2):345-8. DOI: 10.1023/A:1008268930690. View

Tugarinov V, Muhandiram R, Ayed A, Kay L . Four-dimensional NMR spectroscopy of a 723-residue protein: chemical shift assignments and secondary structure of malate synthase g. J Am Chem Soc. 2002; 124(34):10025-35. DOI: 10.1021/ja0205636. View

Delaglio F, Grzesiek S, Vuister G, Zhu G, Pfeifer J, Bax A . NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR. 1995; 6(3):277-93. DOI: 10.1007/BF00197809. View

Rovnyak D, Frueh D, Sastry M, Sun Z, Stern A, Hoch J . Accelerated acquisition of high resolution triple-resonance spectra using non-uniform sampling and maximum entropy reconstruction. J Magn Reson. 2004; 170(1):15-21. DOI: 10.1016/j.jmr.2004.05.016. View

Hwang T, van Zijl P, Garwood M . Broadband adiabatic refocusing without phase distortion. J Magn Reson. 1997; 124(1):250-4. DOI: 10.1006/jmre.1996.1049. View

Vogeli B, Segawa T, Leitz D, Sobol A, Choutko A, Trzesniak D . Exact distances and internal dynamics of perdeuterated ubiquitin from NOE buildups. J Am Chem Soc. 2009; 131(47):17215-25. DOI: 10.1021/ja905366h. View

Wagner G . Prospects for NMR of large proteins. J Biomol NMR. 1993; 3(4):375-85. DOI: 10.1007/BF00176005. View

10.

Lemaster D, Richards F . NMR sequential assignment of Escherichia coli thioredoxin utilizing random fractional deuteriation. Biochemistry. 1988; 27(1):142-50. DOI: 10.1021/bi00401a022. View

11.

Marion D, Driscoll P, Kay L, Wingfield P, Bax A, Gronenborn A . Overcoming the overlap problem in the assignment of 1H NMR spectra of larger proteins by use of three-dimensional heteronuclear 1H-15N Hartmann-Hahn-multiple quantum coherence and nuclear Overhauser-multiple quantum coherence spectroscopy:.... Biochemistry. 1989; 28(15):6150-6. DOI: 10.1021/bi00441a004. View

12.

Koharudin L, Bonvin A, Kaptein R, Boelens R . Use of very long-distance NOEs in a fully deuterated protein: an approach for rapid protein fold determination. J Magn Reson. 2003; 163(2):228-35. DOI: 10.1016/s1090-7807(03)00149-6. View

13.

Clore G, Kay L, Bax A, Gronenborn A . Four-dimensional 13C/13C-edited nuclear Overhauser enhancement spectroscopy of a protein in solution: application to interleukin 1 beta. Biochemistry. 1991; 30(1):12-8. DOI: 10.1021/bi00215a002. View

14.

Vogeli B, Kazemi S, Guntert P, Riek R . Spatial elucidation of motion in proteins by ensemble-based structure calculation using exact NOEs. Nat Struct Mol Biol. 2012; 19(10):1053-7. DOI: 10.1038/nsmb.2355. View

15.

Oschkinat H, Griesinger C, Kraulis P, Sorensen O, Ernst R, Gronenborn A . Three-dimensional NMR spectroscopy of a protein in solution. Nature. 1988; 332(6162):374-6. DOI: 10.1038/332374a0. View

16.

Pervushin K, Riek R, Wider G, Wuthrich K . Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci U S A. 1997; 94(23):12366-71. PMC: 24947. DOI: 10.1073/pnas.94.23.12366. View

17.

Harris C, Millman K, van der Walt S, Gommers R, Virtanen P, Cournapeau D . Array programming with NumPy. Nature. 2020; 585(7825):357-362. PMC: 7759461. DOI: 10.1038/s41586-020-2649-2. View

18.

Ying J, Delaglio F, Torchia D, Bax A . Sparse multidimensional iterative lineshape-enhanced (SMILE) reconstruction of both non-uniformly sampled and conventional NMR data. J Biomol NMR. 2016; 68(2):101-118. PMC: 5438302. DOI: 10.1007/s10858-016-0072-7. View

19.

Tugarinov V, Choy W, Orekhov V, Kay L . Solution NMR-derived global fold of a monomeric 82-kDa enzyme. Proc Natl Acad Sci U S A. 2005; 102(3):622-7. PMC: 545550. DOI: 10.1073/pnas.0407792102. View

20.

Ikura M, Kay L, Bax A . A novel approach for sequential assignment of 1H, 13C, and 15N spectra of proteins: heteronuclear triple-resonance three-dimensional NMR spectroscopy. Application to calmodulin. Biochemistry. 1990; 29(19):4659-67. DOI: 10.1021/bi00471a022. View