On DNA binding peptides and protein-surface interactions
Abstract
The work presented here includes the characterization of the CRE/CREB system, the design of DNA binding peptides and the determination of protein-surface interaction sites by 2D amide exchange spectroscopy. Chapter 1 describes the characterization of the CRE/CREB system. The results include the $\sp1$H NMR assignments of the 14bp CRE DNA sequence as well as that of the peptide sequence corresponding to the CREB dimerization domain. The CREB leucine zipper appears as one continuous helix from residues 1 to 30 in aqueous solution. The CREB bZip module on the other hand, appears to be 43% alpha helical by CD studies. The bZip module exhibits a 20% increase in alpha helical content upon DNA binding. The CREB bZip module displays the same binding specificity as the full protein, however, it exhibits a 200 fold decrease in DNA binding affinity. Analysis by CD and 2D NMR of the CREB bZip module suggests that the distinct structural/functional features reside in the basic region of this peptide. The design and characterization of three putative DNA binding peptides are presented in Chapter 2. These peptides were designed by incorporating the residues from the lac repressor recognition helix into the template peptide sequences. The templates lack any DNA binding activity. The results obtained demonstrated the feasibility of utilizing the GCN4 leucine zipper sequence as a template for this type of work. Chapter 3 describes the use of 2D amide exchange spectroscopy as a probe to determine specific conformational changes occurring upon adsorption of lysozyme to hydrophobic surfaces. Specific changes identified in both turkey and hen lysozyme include the opening of the catalytic cleft concurrently with the exposure of Helix B in the first protein lobe towards the protein surface. This partial unfolding of the protein explains the absence of catalytic activity in surface bound lysozyme.
Degree
Ph.D.
Advisors
Gorenstein, Purdue University.
Subject Area
Biochemistry
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