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The Ohio State University

1. Kumar, Anusha. Exploring packing defects, loop interactions and sequence-fitness landscape in a four-helix bundle protein Rop, by combinatorial and high-throughput approaches.

Degree: PhD, Chemistry, 2019, The Ohio State University

A fundamental challenge of protein engineering is the ability to consistently design proteins that will spontaneously fold into `native-like’ structures. Combinatorial de novo design offers a solution, by limiting the sequence space and helping in understanding sequence-stability relationships. One model system for combinatorial design is Rop, a 63-residue homodimer, whose regulatory function in E. coli is to control plasmid copy number. Rop consists of layers of heptad repeats a-g, with hydrophobic residues occupying a and d positions, forming the hydrophobic core. Interestingly, a break in the pattern results in the surface exposure of an Arg residue at position d and burial of a Phe residue at position e. Understanding the heptad discontinuities and the amino acid preferences at each of the heptad positions in four-helix bundles can aid in the understanding of sequence-structure relationships. We aim to study the conformational preference by means of combinatorial and statistical approaches, and further biophysical characterization.To understand the role of heptad discontinuity in Rop, we designed combinatorial libraries by randomizing Arg55 and Arg55/Phe56 with degenerate codons. A functional cell-based fluorescent screen and conformational stability analysis from thermal and chemical denaturation revealed the role of an electrostatic interaction between the Arg and an Asp present across the dimer interface. Surprisingly, a non-conservative substitution of Arg to Leu is found to increase the stability of the protein by having indistinguishable enthalpy of unfolding as wild type, revealed by Gibbs-Helmholtz analysis. We also found that the R55F56 variants, which have bulky hydrophobes at both positions, are more stable compared to under-packed ones or charged variants, which again have huge enthalpic and entropic penalties. Variants with hydrophobic residues at both 55 and 56 positions are able to compensate for the loss of stability by presumably replacing the electrostatic interaction by increased hydrophobic contacts. Another surprising result is the enhanced stability of a variant (Asp32Arg) that is expected to have repulsive charges at the interaction interface. HSQC data suggest the possibility of altered conformations, and non-canonical heptads for some of these variants revealing the structural plasticity associated with the sequence. To analyze the roles of surface and edge residues in the heptad layers of Rop, we made libraries randomizing each of these heptad positions with degenerate NNK codons. Analysis from the cell based activity screen of these libraries indicate that the surface positional libraries b, c, and f have an overall enrichment of charged residues, with a preference of lysines over arginines on the RNA binding helix 1. The helix 2 could accommodate large aromatic or polar residues for library f, hydrophobic residues for library c, while still charged or polar residues were preferred for library b. The edge positional libraries e and g had an overall enrichment of hydrophobic residues.… Advisors/Committee Members: Magliery, Thomas (Advisor).

Subjects/Keywords: Chemistry

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APA (6th Edition):

Kumar, A. (2019). Exploring packing defects, loop interactions and sequence-fitness landscape in a four-helix bundle protein Rop, by combinatorial and high-throughput approaches. (Doctoral Dissertation). The Ohio State University. Retrieved from http://rave.ohiolink.edu/etdc/view?acc_num=osu1546476128094184

Chicago Manual of Style (16th Edition):

Kumar, Anusha. “Exploring packing defects, loop interactions and sequence-fitness landscape in a four-helix bundle protein Rop, by combinatorial and high-throughput approaches.” 2019. Doctoral Dissertation, The Ohio State University. Accessed September 19, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1546476128094184.

MLA Handbook (7th Edition):

Kumar, Anusha. “Exploring packing defects, loop interactions and sequence-fitness landscape in a four-helix bundle protein Rop, by combinatorial and high-throughput approaches.” 2019. Web. 19 Sep 2019.

Vancouver:

Kumar A. Exploring packing defects, loop interactions and sequence-fitness landscape in a four-helix bundle protein Rop, by combinatorial and high-throughput approaches. [Internet] [Doctoral dissertation]. The Ohio State University; 2019. [cited 2019 Sep 19]. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=osu1546476128094184.

Council of Science Editors:

Kumar A. Exploring packing defects, loop interactions and sequence-fitness landscape in a four-helix bundle protein Rop, by combinatorial and high-throughput approaches. [Doctoral Dissertation]. The Ohio State University; 2019. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=osu1546476128094184

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