High resolution 13C and 1H NMR studies of proteins and peptides

• Main Author: Lian, Lu-Yun
• Published: University of Warwick 1982
• Subjects: 572; QD Chemistry
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.331459

At the magnetic field used in this research (9.4T) many individual 13C resonances of protonated carbon atoms are resolved. To test the applicability of the high-resolution natural abundance 13C NMR method, lysozyme and ribonuclease A are used. The research includes the assignment of many methyl resonances and attempts to answer questions on the behaviour of these side-chains when the proteins are subjected to varying conditions of pD and temperature, and also in the presence of ligands, inhibitors and urea. Ribonuclease A is observed to undergo conformational changes on variation of pD. The region(s) of this protein which are involved consist of many hydrophobic residues such as ile, val and met. Many denaturation processes are effectively in slow exchange at the high magnetic field used. The individual resonances of ribonuclease A which are involved in the predenaturation transitions are identified. They are found to be of distinct types for the two denaturants. In contrast, lysozyme does not show such distinct conformational behaviour below its denaturation temperature. At higher temperatures the main transition from the 'native' to the denatured state of both proteins is two-state. None of the unfolded states of ribonuclease A and lysozyme are random coil. Rather, the unfolded states have definable structures which have hydrophobic bondings and significant, but still restrained, internal flexibility. The high-resolution spectra of ribonuclease A-inhibitor complexes have revealed unprecedented details of the structures of these complexes. Extensive structural changes, including the closure of the active site cleft, together with movements in the hydrophobic regions bordering the cleft, are deduced from the inhibitor-induced shift perturbations of the 13C and 1H NMR resonances of the protein residues. In the assignment work, and the urea denaturation and ligand binding studies, high-resolution 1H NMR is used to complement the 13C NMR technique. A theory relating 13C NMR relaxation parameters to molecular motion at three levels (3—τ) is tested with gramicidin-S and glutathione dimer. The results obtained give information about the conformations and rates of internal librational motions of the two peptides. The '3-τ' model is also applied to some more-limited data on proteins. The low nuclear Overhauser effects are partially explained by this theory.