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Structure and Function of the Mycobacterial Mechanosensitive Channel of Large Conductance, MscL

Citation

Herrera, Nadia (2017) Structure and Function of the Mycobacterial Mechanosensitive Channel of Large Conductance, MscL. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z9JH3J77. http://resolver.caltech.edu/CaltechTHESIS:05162017-130844945

Abstract

MscL is a ubiquitous channel found in bacterial membranes. It provides a protective response to osmotic downshock by opening and closing in response to tension in the membrane. A number of studies have aimed to develop a mechanism for the gating of MscL in E. coli, but structural details describing the process have remained elusive. A few structures of non-conducting states of MscL have been solved using X-ray crystallography, Mycobacterium tuberculosis (Mt) MscL and Staphylococcus aureus (Sa) MscL with a C-terminal domain truncation. In addition, the structure of the E. coli (Ec) MscL C-terminal cytoplasmic domain has been solved.

The goals of the studies presented in this thesis are as follows: (i) capturing a C-terminal domain truncation of MtMscL using X-ray crystallography, and (ii) analyzing the functional regulation of MscL channels in mycobacteria. To achieve the latter goal, we generated a knockout of the mscL gene in a fast-growing mycobacteria species, Mycobacterium smegmatis. This strain was used to analyze the role of MscL in the cell during antibiotic entry. Structural studies of MtMscL are focused on identifying the role of the C-terminal domain by studying a channel with a truncation at the C-terminal domain. The motivation for this goal comes from the structure of SaMscL, which showed that truncation of the C-terminal domain resulted in crystallizing the protein as a tetramer, an alternative oligomeric state to the pentameric state observed for the MtMscL structure. Studies on an MtMscL C-terminal domain truncation aimed to further establish that correlation. This protein was overexpressed in E. coli BL21 DE3 mscL-, purified, and crystallized by sitting drop vapor diffusion. Native crystals diffracted to 6.5 Å, and heavy atom derivative crystals diffracted to 5.8 Å. The structure of the MtMscL C-terminal truncation has been solved, and is presented in this thesis. Our studies on the structure show that the pentameric state of the channel remains intact upon truncation of the C-terminal domain. To analyze the function of our mutant, we utilized patch clamp electrophysiology studies using our expression strain as the giant spheroplast platform. The findings from the electrophysiology studies indicate that MtMscL C-terminal domain truncation results in a channel that has gating tension requirements similar to EcMscL, whereas full-length MtMscL has much higher gating tension requirements than our construct. In addition, the role of MscL in mycobacterial antibiotic susceptibility is being tested in Mycobacterium smegmatis. We have created a strain of M. smegmatis with the mscL gene knocked out, MC2155 mscL- and we have observed that upon deletion of mscL an increase in tolerance to spectinomycin is observed in our knockout strain.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:mechanosensitive channel, MscL, membrane protein structure, x-ray crystallography, patch clamp electrophysiology, mycobacterial allelic exchange, antibiotic resistance
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Biochemistry and Molecular Biophysics
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Rees, Douglas C.
Thesis Committee:
  • Gray, Harry B. (chair)
  • Bjorkman, Pamela J.
  • Newman, Dianne K.
  • Rees, Douglas C.
Defense Date:4 May 2017
Funders:
Funding AgencyGrant Number
Howard Hughes Fellowship ProgramGilliam Fellowship
Howard Hughes Medical InstituteUNSPECIFIED
NIHR01GM084211
Record Number:CaltechTHESIS:05162017-130844945
Persistent URL:http://resolver.caltech.edu/CaltechTHESIS:05162017-130844945
DOI:10.7907/Z9JH3J77
ORCID:
AuthorORCID
Herrera, Nadia0000-0003-4157-9429
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:10175
Collection:CaltechTHESIS
Deposited By: Nadia Herrera
Deposited On:05 Jun 2017 22:48
Last Modified:01 Oct 2018 20:45

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