Studies of internal motions of peptides and proteins in solution by nuclear magnetic resonance spectroscopy
Abstract
Understanding protein function is a primary goal of biological and medical research. The objective of this work is to discover the factors which determine internal motions in protein with the ultimate goal of correlating internal motions with protein function. Despite extensive work that has been performed to date, the details of the relationship between protein dynamics and function are still in question. The principal focus here was to validate and to further develop NMR relaxation techniques as applied to isotopically (13C) enriched peptides and proteins. Internal motional parameters were extracted using the Lipari and Szabo Model-Free approach on a residue-by-residue basis. Specific topics examined here include: (1) measurement of exchange rate and association constant of the monomer-tetramer transformation for wild type melittin and two mutants of melittin enriched with 13 C at specific residues. Different behaviors of monomer-tetramer transition were observed for three types of melittin. Good agreement was found for the association constants obtained by NMR methods, by circular dichroism and by fluorescence measurement. (2) NMR relaxation measurement of tyrosine melittin. No significant difference was found between the dynamics of wild type melittin and the tyrosine mutant of melittin. (3) measurement of internal dynamics of rat intestinal fatty acid binding protein, selectively enriched at alanine backbone a positions and the two ε positions of tyrosine phenol ring with 13C, in the absence and presence of the fatty acids, palmitic acid and oleic acid. Substantial difference was observed in the dynamics of the tyrosine side chains between the ligand bound form of intestinal fatty acid binding protein and the ligand free form. Much less influence of ligand binding was observed on backbone dynamics. These results helped to verify the applicability of NMR relaxation techniques to AX spin system, and to lead to a more comprehensive understanding of the relationship between protein's conformational change and its functional roles.
Degree
Ph.D.
Advisors
Kemple, Purdue University.
Subject Area
Biophysics
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