Nuclear magnetic resonance spectroscopy of high-molecular-weight proteins.
Recent developments in NMR spectroscopy, which include new experiments that increase the lifetimes of NMR signals or that precisely define the orientation of internuclear bond vectors with respect to a common molecular frame, have significantly increased the size of proteins for which quantitative structural and dynamic information can be obtained. These experiments have, in turn, benefited from new labeling strategies that continue to drive the field. The utility of the new methodology is illustrated by considering applications to malate synthase G, a 723 residue enzyme, which is the largest single polypeptide chain for which chemical shift assignments have been obtained to date. New experiments developed specifically to address the complexity and low sensitivity of spectra recorded on this protein are presented. A discussion of the chemical information that is readily available from studies of systems in the 100 kDa mol wt range is included. Prospects for membrane protein structure determination are discussed briefly in the context of an application to an Escherichia coli enzyme, PagP, localized to the outer membrane of gram-negative bacteria.
Tugarinov
Vitali
V
Department of Medical Genetics, University of Toronto, Ontario, Canada M5S 1A8. vitali@pound.med.utoronto.ca
Hwang
Peter M
PM
Kay
Lewis E
LE
D000217:Acyltransferases / Q000737:chemistry; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D008292:Malate Synthase / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D013816:Thermodynamics
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; D011506:Proteins; D000217:Acyltransferases; C468520:PagP protein, E coli; D008292:Malate Synthase