Investigation of the structure and function of SecH, a novel component of the Sec machinery in Escherichia coli

Wynne, Max Alexander (2023). Investigation of the structure and function of SecH, a novel component of the Sec machinery in Escherichia coli. University of Birmingham. Ph.D.

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The Sec machinery translocates proteins across, or inserts proteins into, the cytoplasmic membrane and is responsible for translocation of approximately 20% of all proteins synthesised by the bacterium Escherichia coli. The aim of the work presented in this thesis was to investigate the function, mechanism and structure of a novel component of the Sec machinery, SecH (YecA). SecH contains two structural domains that were identified previously with the aid of bioinformatics: an N-terminal UPF0149 domain and a C-terminal metal binding domain (MBD). The MBD is nearly identical to the C-terminal MBD of the essential ATPase SecA, which mediates the interaction of SecA with the molecular chaperone SecB and with ribosomes. A phylogenetic analysis of the distribution of SecH in different bacterial species presented in this thesis suggested that SecH is strongly co-conserved with SecB. Biochemical and biophysical binding studies indicate that SecH binds to both SecB and ribosomes in a manner that is dependent on the MBD. Structural modelling, size exclusion chromatography and native mass spectrometry indicate that SecH dimerises in solution, and site-specific crosslinking suggests that it forms higher order oligomers in vivo. Copurification experiments indicate that SecH interacts with a broad range of client proteins when overexpressed and these include Sec substrates when expressed in strains with a Sec defect. These results are consistent with previous reports suggesting that SecH has molecular chaperone activity. SecH also copurifies strongly with SecA. Biochemical studies suggest SecH does not modulate the ATPase activity of SecA or increase the rate of ADP dissociation in the absence of SecYEG or substrate protein. However, structural modelling suggests that SecH may directly interact with SecA. Taken together, these results suggest that SecH is a novel component of the Sec machinery that interacts with the ribosome, SecB, SecA and also Sec substrate protein.

Type of Work: Thesis (Doctorates > Ph.D.)
Award Type: Doctorates > Ph.D.
Licence: All rights reserved
College/Faculty: Colleges (2008 onwards) > College of Life & Environmental Sciences
School or Department: School of Biosciences
Funders: Biotechnology and Biological Sciences Research Council
Subjects: Q Science > Q Science (General)
Q Science > QH Natural history > QH301 Biology
Q Science > QH Natural history > QH426 Genetics
Q Science > QR Microbiology


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