Biophysical characterisation of LcrH, a class II chaperone of the type III secretion system

Singh, Sunny Kumar (2015) Biophysical characterisation of LcrH, a class II chaperone of the type III secretion system. Doctoral thesis (PhD), University of Sussex.

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Abstract

The 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, ΔGH2O
= 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 (ΔGH2O = 1.7
kcalmol-1), before cooperatively unfolding to a monomeric denatured state (ΔGH2O =
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.

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Chemistry
Subjects: Q Science > QD Chemistry > QD0241 Organic chemistry > QD0415 Biochemistry
Depositing User: Library Cataloguing
Date Deposited: 20 Nov 2015 15:25
Last Modified: 20 Nov 2015 15:25
URI: http://sro.sussex.ac.uk/id/eprint/58058

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