In the cellular environment, biomolecules assemble in large complexes which can act as molecular machines. Determining the structure of intact assemblies can reveal conformations and inter-molecular interactions that are only present in the context of the full assembly. Solid-state NMR (ssNMR) spectroscopy is a technique suitable for the study of samples with high molecular weight that allows the atomic structure determination of such large protein assemblies under nearly physiological conditions.
This review provides a practical guide for the first steps of studying biological supramolecular assemblies using ssNMR. The production of isotope-labeled samples is achievable via several means, which include recombinant expression, cell-free protein synthesis, extraction of assemblies directly from cells, or even the study of assemblies in whole cells in situ. Specialized isotope labeling schemes greatly facilitate the assignment of chemical shifts and the collection of structural data. Advanced strategies such as mixed, diluted, or segmental subunit labeling offer the possibility to study inter-molecular interfaces.
Detailed and practical considerations are presented with respect to first setting up magicangle spinning (MAS) ssNMR experiments, including the selection of the ssNMR rotor, different methods to best transfer the sample and prepare the rotor, as well as common and robust procedures for the calibration of the instrument. Diagnostic spectra to evaluate the resolution and sensitivity of the sample are presented. Possible improvements that can reduce sample heterogeneity and improve the quality of ssNMR spectra are reviewed.
[NMR paper] Mapping Antibody Epitopes by Solution NMR Spectroscopy: Practical Considerations.
Mapping Antibody Epitopes by Solution NMR Spectroscopy: Practical Considerations.
Related Articles Mapping Antibody Epitopes by Solution NMR Spectroscopy: Practical Considerations.
Methods Mol Biol. 2018;1785:29-51
Authors: Simonelli L, Pedotti M, Bardelli M, Jurt S, Zerbe O, Varani L
Abstract
Identifying an epitope, the region of the antigen in contact with an antibody, is useful in both basic and pharmaceutical research, as well as in vaccine design. Solution NMR spectroscopy is particularly well suited to the residue...
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05-02-2018 11:57 AM
Practical considerations for investigation of protein conformational dynamics by 15 N R 1Ď? relaxation dispersion
Practical considerations for investigation of protein conformational dynamics by 15 N R 1Ď? relaxation dispersion
Abstract
It is becoming increasingly apparent that proteins are not static entities and that their function often critically depends on accurate sampling of multiple conformational states in aqueous solution. Accordingly, the development of methods to study conformational states in proteins beyond their ground-state structure (â??excited statesâ??) has crucial biophysical importance. Here we investigate experimental schemes for optimally...
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03-01-2017 04:13 AM
Structure of the Dimerization Interface in the Mature HIV-1 Capsid Protein Lattice from Solid State NMR of Tubular Assemblies
Structure of the Dimerization Interface in the Mature HIV-1 Capsid Protein Lattice from Solid State NMR of Tubular Assemblies
Marvin J. Bayro and Robert Tycko
http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jacsat/0/jacsat.ahead-of-print/jacs.6b03983/20160628/images/medium/ja-2016-03983c_0006.gif
Journal of the American Chemical Society
DOI: 10.1021/jacs.6b03983
http://feeds.feedburner.com/~ff/acs/jacsat?d=yIl2AUoC8zA
http://feeds.feedburner.com/~r/acs/jacsat/~4/yXorzqET3DQ
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06-29-2016 09:52 AM
[NMR paper] Structure of the dimerization interface in the mature HIV-1 capsid protein lattice from solid state NMR of tubular assemblies.
Structure of the dimerization interface in the mature HIV-1 capsid protein lattice from solid state NMR of tubular assemblies.
http://www.bionmr.com//www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--pubs.acs.org-images-pubmed-acspubs.jpg Related Articles Structure of the dimerization interface in the mature HIV-1 capsid protein lattice from solid state NMR of tubular assemblies.
J Am Chem Soc. 2016 Jun 14;
Authors: Bayro MJ, Tycko R
Abstract
The HIV-1 capsid protein (CA) forms the capsid shell that encloses RNA within a mature...
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06-15-2016 11:12 PM
[NMR paper] Practical considerations over spectral quality in solid state NMR spectroscopy of soluble proteins.
Practical considerations over spectral quality in solid state NMR spectroscopy of soluble proteins.
Practical considerations over spectral quality in solid state NMR spectroscopy of soluble proteins.
J Biomol NMR. 2013 Aug 30;
Authors: Fragai M, Luchinat C, Parigi G, Ravera E
Abstract
Great theoretical and methodological advances are pushing the limits of resolution and sensitivity in solid state NMR (SSNMR). However, sample preparation remains a critical issue for the success of an experiment. The factors affecting spectral quality in...
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08-31-2013 06:56 PM
Site-Directed Methyl Group Labeling as an NMR Probe of Structure and Dynamics in Supra-Molecular Protein Systems: Applications to the Proteasome and to the ClpP Protease.
Site-Directed Methyl Group Labeling as an NMR Probe of Structure and Dynamics in Supra-Molecular Protein Systems: Applications to the Proteasome and to the ClpP Protease.
Site-Directed Methyl Group Labeling as an NMR Probe of Structure and Dynamics in Supra-Molecular Protein Systems: Applications to the Proteasome and to the ClpP Protease.
J Am Chem Soc. 2011 May 11;
Authors: Religa TL, Ruschak AM, Rosenzweig R, Kay LE
Methyl groups are powerful reporters of structure, motion and function in NMR studies of supra-molecular protein assemblies. Their...
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05-12-2011 03:40 PM
Methyl groups as probes of supra-molecular structure, dynamics and function
Methyl groups as probes of supra-molecular structure, dynamics and function
Abstract The development of new protein labeling strategies, along with optimized experiments that exploit the label, have significantly impacted on the types of biochemical problems that can now be addressed by solution NMR spectroscopy. Here we describe how methyl labeling of key residues in a highly deuterated protein background has facilitated studies of the structure, dynamics and interactions of supra-molecular particles. The methyl-labeling approach is briefly reviewed, followed by a summary of...
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01-09-2011 12:46 PM
Protein alignment using cellulose nanocrystals: practical considerations and range of
Abstract Cellulose nanocrystals (CNCs) form liquid crystals in aqueous solution that confer alignment to macromolecules and permit the measurement of residual dipolar couplings. CNCs possess many attractive features as an alignment medium. They are inexpensive, non-toxic, chemically inert, and robust to denaturants and temperature. Despite these advantages, CNCs are seldom employed as an alignment medium and the range of their applicability has not yet been explored. We have re-examined the use of CNCs in biomolecular NMR by analyzing the effects concentration, ionic strength, and...
Structure determination of supra-molecular assemblies by solid-state NMR: Practical considerations #DNPNMR
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