Long peptides for cysteine arylation
Author(s)Evans, Ethan Daniel
Massachusetts Institute of Technology. Department of Chemistry.
Bradley L. Pentelute.
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Biological reactivity is typically carried out by large enzymes. There are few examples of reactive, amino-acid-based polymers shorter than 100 residues in length. Of those that do exist, the majority are very short tags (<15 amino acids). Here, we attempted to first discover peptides roughly 30 amino acids in length that promote a nucleophilic aromatic substitution reaction and then understand the features and properties that emerge. Using the 20 canonical amino acids, there are 20³⁰ different peptide sequences possible in this size realm. To isolate a portion of the space capable of reacting with a perfluoroaromatic small molecule, we performed an mRNA display selection. This uncovered a host of putative reactive peptides with little similarity at the sequence level. The primary isolate (MP01) displayed reactivity confirming the success of the selection and was sensitive to truncation and denaturants. We next set out to study the reactivity of an expanded portion of the isolated peptides and look for shared structural or mechanistic themes. Analyzing an additional 26 peptides with almost no sequence similarity, we discovered diverse levels of reactivity along with sequences capable of undergoing multiple reactions. Using computational structure prediction and circular dichroism spectroscopy we discovered that both mixed alpha-helical and random coil as well as beta-sheet-based reactive peptides existed. Studying their structural properties revealed that many of the peptides undergo significant structural alterations upon reaction with the perfluoroaromatic. Returning to MP01 we studied its mutational tolerance as well as its structural and mechanistic properties. Alanine scanning mutagenesis revealed mutations that diminished reactivity in addition to others that improved its function. Computational structure prediction suggested a mixed helical and random coil structure. Combining the beneficial mutations with insights from modeling initiated an iterative process that ultimately led to a 100-fold improved reaction rate. This sequence (MP01-Gen4) was six mutations different from MPG 1 and was more reactive than any other peptide discovered. MP01-Gen4 displayed flexibility and lacked a defined three-dimensional structure, however, it was significantly more helical than its progenitor. This sequence also displayed structural alterations, becoming more helical in the presence of its small molecule reaction partner, when either covalently reacted or noncovalently interacting.
Thesis: Ph. D. in Biological Chemistry, Massachusetts Institute of Technology, Department of Chemistry, 2018.Cataloged from PDF version of thesis.Includes bibliographical references.
DepartmentMassachusetts Institute of Technology. Department of Chemistry.
Massachusetts Institute of Technology