Dissecting the biophysical mechanisms of phase separation by the myobacterium tuberculosis ABC transporter rv1747
department or school: Medicine, Faculty of
department or school: Biochemistry and Molecular Biology, Department of
data provider: DSpace
degree campus: UBCV
creator: Hui, Lok Tin
issued: 2021-09-17T07:00:00Z
issued: 2021
related degree: Master of Science - MSc
degree grantor: University of British Columbia
description: The ABC transporter Rv1747, which is important for Mycobacterium tuberculosis (Mtb) growth in infected host cells, has a cytoplasmic regulatory module with two phosphothreonine-binding FHA domains joined by a flexible linker with multiple phospho-acceptor threonines (pThr). In vitro, the isolated regulatory module, Rv1747¹⁻³¹⁰, undergoes phosphorylation-enhanced phase separation upon linker phosphorylation by Mtb serine/threonine kinases. Phosphorylation-based phase separation was proposed to result from multivalent intra/intermolecular FHA-pThr interactions. To further dissect the mechanisms of Rv1747 phase separation, I have investigated the factors that influence droplet formation. Visualized with a temperature-controlled microscope, I found that warming phosphorylated Rv1747¹⁻³¹⁰ droplets from 22 oC to 37 oC modestly reduced their size. Studies with Rv1747 truncation fragments and mutants demonstrated that the FHA-1 domain has a predominant role in phosphorylation-enhanced phase separation. Substitution of a key binding residue, S47, in the FHA-1 domain to alanine impaired droplet formation. In contrast, variants with the analogous mutation, S248A, in the FHA-2 domain, or lacking the FHA-2 domain entirely, still underwent phase separation. However, the resulting droplets were gel-like, indicating both FHA domains are required for “regular” droplet formation. The role of the intrinsically disordered linker was also studied. A polypeptide corresponding to the isolated linker did not phase separate. Also, a comparison of Rv1747¹⁻²¹³ (FHA-1 domain and linker) versus Rv1747¹⁻¹¹⁰ (FHA-1 domain only) by NMR spectroscopy indicated the linker residues do not interact with the FHA-1 domain. Substitution of threonine residues in the linker to alanines revealed that T152 is a key residue for the phosphorylation-enhanced phase separation of the regulatory module. I also found that Rv1747¹⁻³¹⁰ treated with PknF in the absence of ATP still underwent phase separation. Based on …
aggregated CHO: https://circle.library.ubc.ca/rest/handle/2429/79794
note: DISSECTING THE BIOPHYSICAL MECHANISMS OF PHASE SEPARATION BY THE MYOBACTERIUM TUBERCULOSIS ABC TRANSPORTER RV1747 by Lok Tin Hui B.Sc., University of Manitoba, 2018 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) September 2021 © Lok Tin Hui, 2021 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Dissecting the biophysical mechanisms of phase separation by the myobacterium tuberculosis ABC transporter rv1747 submitted by Lok Tin Hui in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry and Molecular Biology Examining Committee: Lawrence McIntosh, Department of Biochemistry and Molecular Biology, UBC Supervisor Jörg Gsponer, Department of Biochemistry and Molecular Biology, UBC Supervisory Committee Member Calvin Yip, Department of Biochemistry and Molecular Biology, UBC Supervisory Committee Member Yossef Av-Gay, Department of Microbiology and Immunology, UBC Additional Examiner iii Abstract The ABC transporter Rv1747, which is important for Mycobacterium tuberculosis (Mtb) growth in infected host cells, has a cytoplasmic regulatory module with two phosphothreonine-binding FHA domains joined by a flexible linker with multiple phospho-acceptor threonines (pThr). In vitro, the isolated regulatory module, Rv17471-310, undergoes phosphorylation-enhanced phase separation upon linker phosphorylation by Mtb serine/threonine kinases. Phosphorylation-based phase separation was proposed to result from multivalent intra/intermolecular FHA-pThr interactions. To further dissect the mechanisms of Rv1747 phase separation, I have investigated the factors that influence droplet formation. Visualized with a temperature-controlled microscope, I found th…
has type: Thesis/Dissertation
date issued: 2021-11
is shown at: 10.14288/1.0402339
language: eng
degree discipline: Biochemistry and Molecular Biology
provider: Vancouver : University of British Columbia Library
publisher: University of British Columbia
rights: Attribution-NoDerivatives 4.0 International
rights URI: http://creativecommons.org/licenses/by-nd/4.0/
scholar level: Graduate
contributor: McIntosh, Lawrence
type: Text
identifier URI: http://hdl.handle.net/2429/79794