Master's Theses

Date of Award

2013

Document Type

Thesis

Department

Chemistry

First Advisor

Ronald Koder

Keywords

Supercharging, Protein design, Avian biochemicals

Abstract

De novo protein design offers many interesting prospects both as a means to better understand natural protein dynamics and as a potential resource in biomedical and industrial applications. In this work I describe the modification of a simple, well-characterized heme-binding protein by altering side chain residue identities on the hydrophilic surface of the protein to produce variants with a range of net external charges. These charge modifications had a significant impact on nearly every measurable character of the protein. This work establishes the hard limits of supercharging within our experimental protein scaffold system, demonstrating that excessive positive charge increased the likelihood of amyloid plaque aggregation and excessive negative charge resulted in loss of the alpha-helix character essential to the protein’s heme-binding function. More moderately charged proteins demonstrated that solubility was significantly enhanced in net negatively charged proteins. We were also able to demonstrate a direct relationship between the change in charge of exterior residues and a change in the midpoint potential of the bound heme cofactor. This demonstrated the capacity to “tune” the electric character of bound cofactors without directly modifying the ligand binding site. We further demonstrate the relationship between the high differential between unbound and bound cofactor midpoint potentials and reduced binding affinity. This work supports the notion that covalently bound heme in natural proteins are attached in this manner in order to 4 prevent the heme from dissociating from the protein while still allowing for midpoint potentials greater than 300 mV different from free heme counterparts.

 
 

To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.