Singh, Sunny Kumar.pdf (5.86 MB)
Biophysical characterisation of LcrH, a class II chaperone of the type III secretion system
thesis
posted on 2023-06-08, 23:15 authored by Sunny Kumar SinghThe type three-secretion system (T3SS) is a large and complex protein nano-machine that many gram-negative pathogens employ to infect host cells. A key structure of this machine is a proteinaceous pore that inserts into the target membrane and forms a channel for bacterial toxins to flow from bacteria into the host cell. The pore is mainly formed from two large membrane proteins called “translocators”. Importantly, effective secretion and thus pore formation of the translocators depends on their binding to and being transported by small specialized chaperones after synthesis in the bacterial cytosol. Recent crystal structures have shown these chaperones are formed from modular tetratricopeptide repeats (TPRs). However, each crystal structure produced different homodimeric structures, suggesting flexibility in their topology that may be of importance to function. Given the crucial role of the translocator chaperones, we investigated the conformational stability of the chaperone LcrH (Yersinia pestis). Mutational analysis coupled with analytical ultra-centrifugation and equilibrium chemical denaturations showed that LcrH is a weak and thermodynamically unstable dimer (KD ˜ 15 µM, ?GH2O = 7.4 kcalmol-1). The modular TPR structure of the dimer allows it to readily unfold in a non-cooperative manner to a one-third unfolded dimeric intermediate (?GH2O = 1.7 kcalmol-1), before cooperatively unfolding to a monomeric denatured state (?GH2O = 5.7 kcalmol-1). Thus under physiological conditions the chaperone is able to populate C-terminally unravelled partially folded states, whilst being held together by its dimeric interface. Such ability suggests a “fly-casting” mechanism as a route to binding their far larger translocator cargo.
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148.0Department affiliated with
- Chemistry Theses
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- doctoral
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- phd
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- eng
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University of SussexFull text available
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2015-11-20Usage metrics
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