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Exploring the sequence landscape of the model protein Rop to gain insights into sequence-stability relationship in proteins

Panneerselvam, Nishanthi
http://rave.ohiolink.edu/etdc/view?acc_num=osu1492735031524266

Abstract Details

2017, Doctor of Philosophy, Ohio State University, Biophysics.
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.
Thomas magliery (Advisor)
192 p.
Biochemistry; Biophysics; Molecular Biology

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Citations

  • Panneerselvam, N. (2017). Exploring the sequence landscape of the model protein Rop to gain insights into sequence-stability relationship in proteins [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1492735031524266

    APA Style (7th edition)

  • Panneerselvam, Nishanthi. Exploring the sequence landscape of the model protein Rop to gain insights into sequence-stability relationship in proteins. 2017. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1492735031524266.

    MLA Style (8th edition)

  • Panneerselvam, Nishanthi. "Exploring the sequence landscape of the model protein Rop to gain insights into sequence-stability relationship in proteins." Doctoral dissertation, Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1492735031524266

    Chicago Manual of Style (17th edition)

osu1492735031524266
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This open access ETD is published by The Ohio State University and OhioLINK.