NSU Research Contributions
Title : Characterizing of ZrgB-Permease, the New Zrg Family of ABC Transporters in Vibrio cholerae
Authors : Thathmi Herath Pathiranage; Erik Yukl; Md. Shariful Islam
Abstract : Zinc, as a vital micro-nutrient, plays a crucial role in biological systems. Within the realm of bacteria, the responsible agents for the importation of zinc under conditions of limited zinc availability are the ABC transporters. The microorganism known as Vibrio cholerae is comprised of two distinct gene clusters, namely znuABC and zrgABCDE. These clusters are under the control of a putative regulator known as Zur. Every system is comprised of a membrane-bound permease and a cytoplasmic ATPase. These components rely on a periplasmic solute binding protein (SBP) that exhibits selectivity in binding substrates and transporting them to the permease. Nevertheless, it is worth noting that there is a conspicuous absence of established frameworks pertaining to the Zn-specific permeases or ATPases associated with Zrg, an operon of ABC transporters. In the current study, the proteins ZrgB and ZrgC, specifically the permease and ATPase, were concurrently expressed as fusion proteins tagged with green fluorescent protein (GFP). These fusion proteins were subsequently solubilized in the anionic detergent DDM. The purification of protein was achieved through the utilization of nanobody-coupled cyanogen bromide resin (CNBR), followed by the subsequent cleavage of the protein from the resin using PPX protease. Nevertheless, the SDS-PAGE data unveiled the exclusive expression of ZrgB, while the presence of ZrgC remained undetected. The mass spectrometry analysis has provided evidence that ZrgB encompasses a repertoire of 15 distinct peptides. Furthermore, the utilization of inductively coupled plasma optical emission spectroscopy has yielded valuable insights, demonstrating the binding of ZrgB to a quantity of 1.05 equivalents of zinc, whereas dynamic light scattering revealed ZrgB's monodispersity. Furthermore, based on the assumption of a spherical shape, the predicted particle size of ZrgB has been estimated to be 20000g/mol. Additionally, the ZrgB protein has been successfully incorporated into a peptidisc structure using an on-column reconstitution technique. This method was employed to achieve solubilization of ZrgB in a detergent-free buffer, thereby promoting a detergent-free environment. The findings exhibit encouraging potential in the characterization of ZrgB, prompting further investigations aimed at unraveling the intricate structural model governing the interaction between ZrgA, a substrate-binding protein, and its corresponding permease, ZrgB. In summary, these studies will delve into the conformational alterations that facilitate the transportation of zinc ions
| Journal : | Volume : | Year : 2024 | Issue : |
| Pages : | City : | Edition : | Editors : |
| Publisher : Elsevier | ISBN : | Book : | Chapter : |
| Proceeding Title : The American Society for Biochemistry and Molecular Biology | Institution : | Issuer : | Number : |
