Theses and Dissertations

Date of Award

5-2023

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biochemistry and Molecular Biology

First Advisor

Dr. Yonghong Zhang

Second Advisor

Dr. Upal Roy

Third Advisor

Dr. Arnulfo Mar

Abstract

Helicobacter pylori (H. pylori) is a Gram-negative bacterium that colonizes the human gastric mucosa and is associated with peptic ulcer disease and gastric cancer. Bacterial protein synthesis is a fundamental metabolic process occurring in all bacteria and is a validated target of antibiotics; however, the precise structural mechanism of H. pylori protein biosynthesis remains unknown. Protein synthesis contains four stages, and translational initiation is the most important and highly regulated critical step, involving three initiation factors (IF1, IF2, IF3) and other key components. IF1 plays a vital role in regulating this step by binding to the 30S ribosomal subunit cooperatively with other initial components. However, H. pylori’s IF1 structure is unknown. Using a computational model, this study investigated the interaction between H. pylori initiation factor IF1 and the 30S ribosomal subunit. Hp-IF1 was subcloned into pET24b vector for constructing recombinant DNA plasmid- pET24b-Hp-IF1 DNA plasmid. The recombinant DNA plasmid was used to express the Hp-IF1 protein by the E. coli expression system. The proteins were purified by column chromatography, which were used for NMR structural analysis. The Hp-IF1 protein structure was predicted and used for building the complex model binding with the 30S subunit. We elucidated the structure of the IF1-bound 30S complex, revealing the importance of a short α-helix in IF1 for ribosomal binding and function. A peptide derived from this α-helix Hp-IF1 was designed and tested by MIC assay showing broad-spectrum activity against Gram-negative and positive bacteria, including H. pylori. The peptide shows a low cytotoxicity effect against human embryonic kidney (HEK-293) cells. These results suggest Hp-IF1 peptide as a potential compound for the development of new antimicrobial agents.

Comments

Copyright 2023 Asif Shahriar. All Rights Reserved.

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