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Hossein Thesis Final.pdf (12.41 MB)

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Using Experimental and Computational Methods to Study Loop Mutations in a Four-bundle Helix Protein

Ashrafian, Hossein
http://rave.ohiolink.edu/etdc/view?acc_num=osu1595370458757093

Abstract Details

2020, Master of Science, Ohio State University, Chemistry.
Rop protein is a four-bundle dimeric protein that assists RNAI to form a kissing complex with RNAII. By engagement in the kissing complex, the DNA polymerase will fail to replicate from the ColE1 origin and consequently, the copy number of the ColE1 plasmids will be downregulated. Rop’s monomer is a helix-turn-helix structure with a tight turn containing a Schellman motif. We have studied the stability and activity of the Rop protein with different mutations in the loop section of the protein, residues 29-32, Leu-Asp-Ala-Asp (LDAD) in wild-type. The Arg55 residue appears to make an ionic contact with Asp32 in the wild-type. We engineered four variants, LDAG, R55Q_LDAN, LGGAD, and R55Q_LGDAD and compared their stability and activity with AV_Rop, as the native Rop with LDAD sequence in its loop region. To measure their stability, first, all these four variants were cloned into a T7 expression vector, and after expression and purification, their thermal stability was measured by CD thermal scanning. The result showed that the variant R55Q_LGDAD was the most stable variant. LGGAD and LDAG were more stable than wild-type one but less than R55Q_LGDAD. Finally, the variant R55Q_LDAN was the least stable variant. To measure the activity of the variants, all the variants were cloned into p15a vector and using green fluorescent protein (GFP) expression from a ColE1 plasmid, their activity was measured. We also ran a 1 ns MD simulation to validate our simulation system for future longer runs. We have plan to calculate RMSD, RMSF, DSSP, SASA, HB, Ramachandran plot, and free energy surface of all variants under longer runs.
Thomas Magliery (Advisor)
Rafael Brüschweiler (Committee Member)
99 p.
Biochemistry; Chemistry
protein stability, protein engineering, Rop protein, rational design

Recommended Citations

Citations

  • Ashrafian, H. (2020). Using Experimental and Computational Methods to Study Loop Mutations in a Four-bundle Helix Protein [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1595370458757093

    APA Style (7th edition)

  • Ashrafian, Hossein. Using Experimental and Computational Methods to Study Loop Mutations in a Four-bundle Helix Protein. 2020. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1595370458757093.

    MLA Style (8th edition)

  • Ashrafian, Hossein. "Using Experimental and Computational Methods to Study Loop Mutations in a Four-bundle Helix Protein." Master's thesis, Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1595370458757093

    Chicago Manual of Style (17th edition)

osu1595370458757093
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This open access ETD is published by The Ohio State University and OhioLINK.