Elucidating Loop and Core Tolerance and Dynamics in Alpha-Helical Proteins
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Date
2017-05
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The Ohio State University
Abstract
Linking the sequence of a protein to its stability and function and being able to predict how a protein will fold from its sequence has long been a goal of biochemical researchers. A particular region of interest in a protein is its core. The composition of a protein core, among other elements, will determine and drive the topological formation of the entire molecule. Previous work has been done concerning how mutations within the core of Rop, an alpha-helical protein composed of 63 amino acids that forms an anti-parallel homodimer, changes the structure and stability. Through characterization of a set of Rop variants, we observed that repacking Rop’s core leads to variable outcomes. In general, the protein core is relatively intolerant to mutation, but how resilient are the loops of proteins to mutation, and what sequence of amino acids are found in the loops of the most active Rop variants? Using Rop, we can model how randomization of a loop region alters the biophysical characteristics of a protein. First, we have randomized the canonical four residues of Rop’s loop using degenerate codons to create a protein library in both wild-type Rop and a Rop variant engineered to remove a potentially stabilizing ionic interaction between a loop residue of one monomer and a residue near the C terminus of the second monomer. Two other protein libraries were also created using the same two Rop constructs but with an additional fifth residue in the loop. These four protein libraries are being subjected to a cell-based screen that connects phenotype to activity within the cell. Fluorescent ‘active’ constructs will be chosen for High Throughput Thermal Scanning, or HTTS, for final library characterization of the stability of selected variants. Additionally, through an enrichment process, we will observe the composition of the most favorable Rop loops. Future studies will include further characterization of a select few interesting variants by thermal and chemical denaturation and NMR. From these data, we will draw conclusions about how tolerant Rop’s loop is to mutation. These conclusions may lend insight into the complicated relationship between sequence and fold and provide useful knowledge for therapeutic development and industrial processes.
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Keywords
Protein engineering, Loop, Hydrophobic core, Protein stability