Title

Designing Proteins and Peptides for Catalysis, Biophysical Studies and Therapeutic Applications

Date of Award

May 2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Advisor(s)

Ivan V. Korendovych

Second Advisor

Kevin Sweder

Keywords

Biophysical studies, Catalysts, Peptides, Proteins, Therapeutic application

Subject Categories

Physical Sciences and Mathematics

Abstract

Enzymes are large biomolecules which catalyze complex reactions with high selectivity and efficiency. The function of an enzyme depends on the unique three-dimensional structure dictated by the amino acid sequence. Understanding the evolutionary origin of enzyme’s structure and function is pivotal for designing proteins/peptides with novel or enhanced function. Extensive research efforts in this field have led to successful strategies which combine sophisticated computational algorithm and high-throughput combinatorial techniques. These strategies have been used to develop peptides and proteins for biocatalysis, antibody therapeutics and drug delivery applications etc. In this work, we used a minimalistic approach with high-throughput screening to design efficient peptides and proteins with novel functions.

In the initial study, we designed a library of short 7-residue peptides that self-assembled and catalyzed ester hydrolysis in presence of ZnII with activity at par with the natural enzyme by weight. Furthermore, self-assembly of these peptides in presence of CuII allowed us to create a catalyst for oxygen activation. Thus, we demonstrated that self-assembly of peptides can present an excellent tool to design catalyst for difficult chemical transformations. More importantly, this approach enables screening of multiple co-ordination spheres, simply by mixing different peptides. The ease with which we were able to develop these catalysts suggest that short peptide-assemblies might have served as intermediates in enzyme evolution.

Additionally, we applied a minimalistic approach to design a catalyst for selective post translational modification. Strategic incorporation of single His residue at position 144 in a non-enzymatic eukaryotic protein calmodulin (CaM) introduced hydrolytic and acylase activity into the protein. The designed allosterically regulated acylase was capable of site selective acylation of Lys residues in CaM-binding peptides involved in regulation of various cellular processes. Moreover, this acylase can serve as a unique chemical biology tool to identify CaM-binding domains and elucidate structural interactions between CaM and its binding partner.

In this work, we also demonstrated that introduction of an unnatural Trp analog, AzAla into peptides/proteins can allow monitoring of protein-protein interaction, protein folding and dynamics. The weak environmental dependence along with the sensitivity to weak intrinsic quenchers makes AzAla a unique fluorescent probe for such studies. Substitution of AzAla in place of Trp in M2 channel of influenza A enabled us to monitor the protonation state of His residues with minimum perturbation to function. Moreover, with the availability of technology to introduce AzAla translationally into proteins, we envision that AzAla will gain wide utility in biophysical studies.

Lastly, we utilized a rational design approach to develop a peptide microbicide candidate for prevention of sexual transmission of HIV. HIV is a great public health challenge worldwide and effective strategies are required to stop the spread of this pandemic. Current antiretroviral therapy is successful; however, the approach is also susceptible to developing drug resistance. Recently, it was demonstrated that amyloid fibrils formed in semen called semen-induced enhancer of virus infection (SEVI) increased HIV infectivity by several-fold in vivo. In this work, we designed a peptide E-FK16 that selectively interacts with SEVI. Using E-FK16, we created a randomized library which would be screened using phage display to identify a peptide candidate with greater ability to interact with SEVI and thus prevent HIV transmission through SEVI. SEVI presents a unique target to develop microbicide for prevention of HIV transmission as this strategy does not target the viral machinery and hence it would be difficult for the virion to develop resistance.

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