Main content

Exploring Pepsin’s Alkaline Instability via Bioinformatic Analysis and a Rationale Protein Design Approach

Show full item record

Title: Exploring Pepsin’s Alkaline Instability via Bioinformatic Analysis and a Rationale Protein Design Approach
Author: Grahame, Douglas Stuart Alexander
Department: Department of Food Science
Program: Food Science
Advisor: Yada, Rickey Y
Abstract: Residues and motifs defining to the alkaline instability of pepsin and alkaline stability of renin have, as of yet not been identified. To accomplish said task the present study utilized literature and a comparative bioinformatic analysis to generate structural and functional information regarding protein stability, protein folding, and mutational stability related to alkaline stability. Site-directed mutagenesis was employed to generate 15 mutants in the N-terminal domain expected to increase the alkaline stability of pepsin. Mutants D159L and D60A increased alkaline activity (P≤0.05) whereas E4V and H53F were found to retain native structure at elevated pH levels (P≤0.05). Alleviating charge – charge repulsion of carboxyl groups of interest was insufficient to increase the alkaline stability of pepsin. The importance of the β-barrel was highlighted as 92% of the stabilizing residues identified by SRide in the N-terminal lobe were located in β-structure of pepsin and renin. The comparative bioinformatic analysis identified structure and sequence differences between pepsin and renin in β-strands and turn/loop regions of the N-terminal lobe known to play a role in the alkaline denaturation of pepsin. Differences in residues promoting acidic or alkaline stability were identified in pepsin and renin. Flexibility and rigidity differences were identified where renin was found to be a more rigid protein with specific areas of flexibility whereas pepsin has a higher level of general flexibility. In summary, alkaline activity and stability was improved by reducing electrostatic repulsion of Asp159. The ability of Asp159 to improve activity (P≤0.05) denotes electrostatics do play some role in alkaline denaturation. However, overall alkaline activity and structural stability was not dramatically improved for any of the mutants generated. Thus, alkaline stabilization of pepsin will require more than the point-mutation reduction of electrostatic repulsions. The presence of stabilizing residues within β-barrel structure implicates the hydrogen-bonding network of the β-barrel as important in defining stability. Finally, flexibility differences and amino acid composition indicate that the increased rigidity of β-sheets, the ψ-loop, and turn regions likely play a defining role in the alkaline stability difference between renin and pepsin, and that stabilization occurs at a motif level rather than at a residue level.
URI: http://hdl.handle.net/10214/10286
Date: 2017-03
Rights: Attribution-NonCommercial-NoDerivs 2.5 Canada
Terms of Use: All items in the Atrium are protected by copyright with all rights reserved unless otherwise indicated.


Files in this item

Files Size Format View Description
Grahame_Douglas_201703_PhD.pdf 16.23Mb PDF View/Open Thesis

This item appears in the following Collection(s)

Show full item record

Attribution-NonCommercial-NoDerivs 2.5 Canada Except where otherwise noted, this item's license is described as Attribution-NonCommercial-NoDerivs 2.5 Canada