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Author
Title Exploring the sequence landscape of the model protein Rop to gain insights into sequence-stability relationship in proteins
URL
Publication Date
Degree PhD
Discipline/Department Biophysics
Degree Level doctoral
University/Publisher The Ohio State University
Abstract Surface residues and surface electrostatics play an active role in maintaining protein stability. A combinatorial library randomizing five surface positions in Rop to NNK (K=G or T) codons was constructed. Interestingly, the consensus sequence had positively charged residues instead of negatively charged residues present in wild-type. Specifically, lysines were found more than arginines. To delve into this, all these five positions were individually mutated to Lys and Arg and four poly mutants were made by mutating to multiple lysines and arginines. Most of the point mutants were found to be active by Rop screen. All mutants were well folded as seen by circular dichroism studies. An important result was that most of the mutants were more thermally stable than the Cys-free wild-type scaffold AV-Rop. Thermodynamic parameters were found using Gibbs-Helmholtz analysis and the entropic change was higher for most mutants. Solubility was affected more in the poly mutants than in the point mutants. HSQC on selected variants revealed that the least stable mutant and poly mutants had more shifted peaks compared to the most stable mutant. No considerable differences were found between Lys and Arg mutants. Poly mutants had varied effects on activity and solubility compared to point mutants.If all the possible point mutants in a protein are subject to moderate selection for activity or function, high-throughput sequencing can measure the relative abundance of mutants in a bulk competition experiment which in turn gives the relative fitness of each mutant. This project involves making all possible point mutants of Rop by constructing 20 member positional libraries individually in 62 positions of Rop. Preliminary data includes proof-of-principle selection experiments in three positions of Rop (F14, D30 and D43). Variants were enriched 1,000 fold for activity by performing six rounds of growth based selection. Likewise, we are in the process of constructing 62 positional libraries which will be combined together in the selection screen, subject to enrichment and then sequenced by high-throughput methods to give the sequence landscape. Analyzing the sequence/fitness landscape of Rop will yield each mutant’s competitive advantage/disadvantage.
Subjects/Keywords Biochemistry; Biophysics; Molecular Biology
Contributors magliery, Thomas (Advisor)
Language en
Rights unrestricted ; This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.
Country of Publication us
Format application/pdf
Record ID oai:etd.ohiolink.edu:osu1492735031524266
Repository ohiolink
Date Retrieved
Date Indexed 2019-05-21
Grantor The Ohio State University

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