Full metadata record
| DC Field | Value | Language |
|---|---|---|
| dc.contributor | Department of Applied Biology and Chemical Technology | en_US |
| dc.contributor.advisor | Yao, Zhong-ping (ABCT) | en_US |
| dc.creator | Huang, Liwen | - |
| dc.identifier.uri | https://theses.lib.polyu.edu.hk/handle/200/10736 | - |
| dc.language | English | en_US |
| dc.publisher | Hong Kong Polytechnic University | en_US |
| dc.rights | All rights reserved | en_US |
| dc.title | Investigation of interactions between β-lactamases and β-lactamase inhibitory proteins (BLIPs) using mass spectrometric approaches | en_US |
| dcterms.abstract | β-Lactamase inhibitory protein (BLIP) can effectively inactivate a variety of class A β-lactamases, but with very different degrees of potency. Understanding the roles of BLIP in class A β-lactamases inhibition can provide insights for novel inhibitor design. However, this problem was poorly solved based on the static structures obtained by X-ray crystallography. In this study, ion mobility mass spectrometry, hydrogen deuterium exchange mass spectrometry and molecular dynamics simulation revealed the conformational dynamics of three class A β-lactamases with varying inhibition efficiency by BLIP. A more extended conformation of PC1 were shown compared to TEM1 and SHV1. Localized dynamics showed differences in several important loop regions, i.e., the protruding loops, H10 loops, Ω loops and SDN loops of the three β-lactamases. Upon binding with BLIP, these loops cooperatively rearranged to enhance the bindings and inactivate the catalytic sites. Unfavorable changes in conformational dynamics were found in the protruding loops of SHV1 and PC1. Intriguingly, single mutations on BLIP could compensate the unfavored changes in this region, and thus exhibited enhanced inhibition towards SHV1 and PC1. Additionally, the H10 region was revealed as an important allosteric site that could modulate the inhibition of class A β-lactamases. It was suggested that the rigid protruding loop and flexible H10 region might be determinants for the effective inhibition of TEM1. Our findings provided unique and explicit insights into the conformational dynamics of β-lactamases and their bindings with BLIP. The wide range of ability to bind the β-lactamases is also still a puzzle. The structural determinants to the significant enhancement of BLIP with single mutations are poorly understood yet. To further the understanding of these inhibitory interactions, we investigated the conformational dynamics of BLIP and three enhanced mutants (Y50A, E73M and K74G) upon their bindings to three clinically prevalent class A β-lactamases (TEM1, SHV1 and PC1), with varying potency between subnanomolar and micromolar. Hydrogen deuterium exchange mass spectrometry revealed that the flexibility of interdomain region was significantly suppressed upon the strong binding to TEM1 β-lactamases. By contrast, such change was not significant upon the weak bindings to SHV1 or PC1. Single mutations E73M and K74G on the interdomain region with improved potencies towards SHV1 and PC1, respectively, showed significantly improved flexibility of the interdomain region compared to the wild type. In contrast, more rigid structure of the β-hairpin loop 135-145 was observed upon individual mutation on BLIP. These results indicated that higher flexibility of the interdomain linker while more rigid interfacial β-hairpin loop 135-145 could be desirable for enhanced inhibitory bindings. Molecular dynamics simulation of these mutations of BLIP exhibited significant changes in the flexibility of the β-hairpin loop 46-51 and the distant β1'-β2' loop 115-125 compared to the wild type. This study can be extended to other β-lactamases of interest, especially the inhibitor resistant β-lactamases. Together, these findings provided a general guideline for studying the interactions between β-lactamases and inhibitory proteins. Unique insights and deep understandings were obtained for the design of BLIP-derived inhibitors. | en_US |
| dcterms.extent | xxii, 217 pages : color illustrations | en_US |
| dcterms.isPartOf | PolyU Electronic Theses | en_US |
| dcterms.issued | 2020 | en_US |
| dcterms.educationalLevel | Ph.D. | en_US |
| dcterms.educationalLevel | All Doctorate | en_US |
| dcterms.LCSH | Mass spectrometry | en_US |
| dcterms.LCSH | Proteins -- Analysis | en_US |
| dcterms.LCSH | Beta lactamases | en_US |
| dcterms.LCSH | Hong Kong Polytechnic University -- Dissertations | en_US |
| dcterms.accessRights | open access | en_US |
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