Expanding the Role of Proteins and Peptides for Biochemical and Therapeutic Applications
Date of Award
Doctor of Philosophy (PhD)
Ivan V. Korendovych
Physical Sciences and Mathematics
Billions of years of evolution have resulted in natural enzymes that can catalyze complex chemical reactions in living organisms with high selectivity and efficiency. The ability to design enzymes for a specific purpose is of great practical interest and has garnered a lot of attention in recent decades. This has led to the development of various approaches, which combine computational methods with combinatorial techniques for designing enzymes incorporating novel or enhanced functions. Such designed enzymes have been successfully utilized in biocatalysis and therapeutic applications. In this work, we have employed a minimalistic approach to design proteins and peptides with novel functions.
We have shown the application of a designed catalyst for selective post-translational modification. Strategic incorporation of a single histidine residue at position 144 in a non-enzymatic calmodulin (CaM), enables the protein to catalyze ester hydrolysis and acyl-transfer reactions. Allosterically regulated CaM M144H is capable of site selective acylation of lysine residues in CaM-binding peptides. CaM is involved in the regulation of a number of biological processes and therefore, this chemical biology tool can be utilized to identify novel CaM-binding partners as well as provide structural insights into their interactions with CaM.
In an extension of the above work, we have demonstrated the application of CaM M144H as a chemical probe to label CaM binding proteins in cell lysate using an unnatural tag that can be easily identified using mass spectrometry. From the cell lysate screen, we selected the potential CaM-binding domains of various proteins, previously uncharacterized to bind CaM, and further studied their interaction using isothermal titration calorimetry. By employing this method, we have identified novel CaM targets without using any complicated methodology or complex library generation.
Additionally, we have studied the tyrosyl radical (Y•) formation in ribonucleotide reductases (RNRs), an essential enzyme that catalyzes the conversion of ribonucleotides to deoxyribonucleotides. Using mutagenesis and spectroscopic analysis, we have studied the assembly of metallocofactor and radical formation in class Ib RNR of human pathogens belonging to the Streptococci family. Our investigations lay a foundation to elucidate the pathway of MnIII2-Y• metallocofactor assembly, blocking of which can be an attractive novel antimicrobial target.
In this work, we also studied the synergistic non-covalent interactions and effects of chirality in short self-assembling catalytic peptides. By introducing a linker in these peptides to form β-hairpin loops, we have demonstrated the propagation and improvement of initial catalytic activity in complex molecules. These studies are important for the development of more active catalysts in the future.
Lastly, using a rational design approach we have developed peptide candidates for the prevention of sexual transmission of HIV. Fragments of PAP and SEM proteins found in semen are known to form amyloid fibrils, that can increase the infectivity of HIV over several orders of magnitude. We have designed peptides capable of selectively interacting with these fibrils. Using the phage display method, we have also screened for peptide candidates with enhanced ability to interact and block PAP fibrils. This work can lead to the development of a novel semen amyloid antagonist capable of inhibiting the attachment of HIV to the host cell and therefore, reduce HIV infectivity through sexual transmission.
SURFACE provides description only. Full text may be available to ProQuest subscribers. Please ask your Librarian for assistance.
Jayachandran, Megha, "Expanding the Role of Proteins and Peptides for Biochemical and Therapeutic Applications" (2020). Dissertations - ALL. 1226.