Theoretical and practical application of exchange transferred nuclear Overhauser effects in peptide structure determination and preliminary studies on band 3 inhibition of two glycolytic enzymes
Abstract
This study explored the use of the exchange-transferred NOE (etNOE) in structure determination of peptide:protein complexes both to determine peptide structure and to examine the limitations that should be expected when using this technique. Chapter 1 provides an overview of NMR theory and the application of the etNOE when solving bound ligand structures. Chapter 2 describes eight methods to build a complex for a band 3 peptide and aldolase based on experimental restraints for the peptide and the enzyme crystal structure. An automated process was found to be as successful as human intervention in producing a plausible complex, and the automated process found an alternative binding arrangement for the peptide not determined from direct user manipulation. Chapter 5 continued an examination of this system with work intended to resolve counter-intuitive kinetic studies of band 3 with two glycolytic enzymes, aldolase and G3PDH. Difficulty in producing two fragments of band 3 prevented further work in this area. Chapter 3 explores five different restrained molecular dynamics simulation procedures using theoretically determined etNOE restraints for two peptides derived from a crystallographically determined complex. No significant advantage was found for any of the approaches. However, the peptide bound in an extended conformation was not calculated to have any long-distance restraints for half of the sequence, and this peptide was determined with much less precision and accuracy than a second peptide which was primarily alpha-helical. Chapter 4 examines the effects of intermolecular spin diffusion when determining bound ligand structures using nineteen high-resolution X-ray complexes and calculating the expected inter- and intramolecular spin diffusion effects for each. Intermolecular spin diffusion was found, but with effects comparable in size to that seen for intramolecular spin diffusion. As a result, standard practices for resolving spin diffusion in proteins were found to be equally applicable for structure determination of bound ligands. A discussion of the NMR R factor is also presented, as is a comparison between structures selected based on R factor or low NOE energy. It was found that the R factor analysis provided structures with slightly better accuracy and precision for two related peptides.
Degree
Ph.D.
Advisors
Post, Purdue University.
Subject Area
Biophysics|Pharmacology
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