High-resolution NMR spectroscopy and X-ray diffraction are the only techniques providing atomic-level structural information on proteins. In addition, NMR has the unique capability of allowing researchers to investigate the internal dynamics of the polypeptide in solution over a wide range of timescales. By coupling structural and dynamic aspects, NMR spectroscopy thus affords a complete picture of the behavior of proteins. We start by describing so-called multidimensional NMR experiments, which are performed on samples enriched in 15N and 13C stable isotopes (and possibly 2H, for larger proteins) to determine the frequency of resonances of each individual nucleus in the protein. This first step is necessary to enable all further studies on structure and dynamics. To structurally characterize a protein, a number of conformational restraints must be collected that typically consist mainly of upper limits on hydrogen-hydrogen distances and dihedral angle restraints; these can be supplemented with residual dipolar couplings. The methods for the collection of these restraints and the computational approaches that exploit them to obtain an energy-optimized structure are explained in some detail. Structure calculation methods relying exclusively on chemical shift information are also mentioned. After a protein structure has been determined, it has to be thoroughly validated before making it available to the scientific community. This step is carried out through a combination of evaluation against experimental data and comparison to standard stereochemical/geometric features. Finally, we address the characterization of protein dynamics based on NMR relaxation measurements as well as on other experimental approaches on various timescales. Protein dynamics can be investigated independently of or in parallel with structure determination projects.

Determination of protein structure and dynamics / L. Ferella; A. Rosato; P. Turano. - STAMPA. - (2012), pp. 53-94. [10.1002/9783527644506.ch4]

Determination of protein structure and dynamics

FERELLA, LUCIO;ROSATO, ANTONIO;TURANO, PAOLA
2012

Abstract

High-resolution NMR spectroscopy and X-ray diffraction are the only techniques providing atomic-level structural information on proteins. In addition, NMR has the unique capability of allowing researchers to investigate the internal dynamics of the polypeptide in solution over a wide range of timescales. By coupling structural and dynamic aspects, NMR spectroscopy thus affords a complete picture of the behavior of proteins. We start by describing so-called multidimensional NMR experiments, which are performed on samples enriched in 15N and 13C stable isotopes (and possibly 2H, for larger proteins) to determine the frequency of resonances of each individual nucleus in the protein. This first step is necessary to enable all further studies on structure and dynamics. To structurally characterize a protein, a number of conformational restraints must be collected that typically consist mainly of upper limits on hydrogen-hydrogen distances and dihedral angle restraints; these can be supplemented with residual dipolar couplings. The methods for the collection of these restraints and the computational approaches that exploit them to obtain an energy-optimized structure are explained in some detail. Structure calculation methods relying exclusively on chemical shift information are also mentioned. After a protein structure has been determined, it has to be thoroughly validated before making it available to the scientific community. This step is carried out through a combination of evaluation against experimental data and comparison to standard stereochemical/geometric features. Finally, we address the characterization of protein dynamics based on NMR relaxation measurements as well as on other experimental approaches on various timescales. Protein dynamics can be investigated independently of or in parallel with structure determination projects.
2012
9783527328505
NMR of biomolecules. Towards mechanistic systems biology
53
94
L. Ferella; A. Rosato; P. Turano
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Utilizza questo identificatore per citare o creare un link a questa risorsa: https://hdl.handle.net/2158/654268
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