Biochemical and structural studies of a band 3 peptide with glycolytic enzymes, four-dimensional energy embedding, accuracy of bound peptide structures determined by exchange transferred nuclear Overhauser data, and NMR studies of a virally encoded fungal toxin

Elan Zohar Eisenmesser, Purdue University

Abstract

The N-terminal cytoplasmic domain of the transmembrane protein band 3 is known to bind and inhibit several glycolytic enzymes. Chapter 1 serves as a review of this domain. Chapters 2 and 3 contain the biochemical and structural studies, respectively, of a synthetic peptide derived from this N-terminal cytoplasmic domain with the glycolytic enzymes G3PDH and aldolase. The implementation of four dimensional energy embedding to both the minimization and molecular dynamics algorithms of the program CHARMM is described in Chapter 4. This technique uses artificial spatial dimensions to surmount local minima in conformational space. The application of 4D molecular dynamics in free energy simulation methods is shown to successfully predict the free energy of solvation for several small molecules into bulk water laying the ground work for future fire energy calculations such as ligand-protein interactions (i.e. dissociation constants). The accuracy of bound peptide structures determined from exchange transferred nuclear Overhauser (etNOE) data is addressed in Chapter 5. This is the NMR technique used to determine the B3P structure bound to G3PDH (Chapter 3) as well as its structure previously determined in this lab bound to aldolase. Though etNOE interactions have been used extensively for determining bound peptide structures for over a decade there has not yet been a model study addressing the question of accuracy. Chapter 6, the final chapter of this thesis, includes results from a number of NMR experiments on the KP4 toxin. The Xray crystal structure of this virally encoded toxin has recently been determined to 1.4 Å, yet there remain several questions that NMR has already begun to answer. The diffusion coefficient determined by NMR is consistent with a monomer. A number of 3D-NMR experiments were done with the aim of assigning the resonances of the KP4 toxin which would allow for the solution structure determination as well as binding studies to the toxin receptor. Since these 3D experiments also provide structural information, such as &phis; and χ1 angles, the NMR derived parameters for those residues already assigned were compared to the crystal structure and were found to be similar.

Degree

Ph.D.

Advisors

Post, Purdue University.

Subject Area

Biophysics

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