Abstract
Biological processes involve complex spatial and temporal coordination between proteinsand other biomolecules, important for a multitude of cellular functions. One of the major
challenges in cell and structural biology is the ability to capture information about the
interactions between the macromolecular complexes that underlie important cellular
processes. Defining the molecular basis of these interactions is crucial to understanding
when they are dysregulated in disease.
Protein complexes are not static structures and are subject to dynamic changes. In order
to understand their function at a molecular level, these motions must be considered.
Investigating the structure, interactions and dynamics of macromolecular protein
assemblies remains a challenge for well-established biophysical techniques. Recent
advances in structural mass spectrometry (MS) techniques has enabled a plethora of
information to be obtained. MS is a powerful tool that can be combined with other
biophysical, biochemical and computational approaches to provide invaluable insights
into the molecular mechanism of proteins and their complexes.
This thesis aims to study the structure, dynamics and interactions of three large
macromolecular assemblies (soluble and membrane proteins) using structural MS.
Specifically, the structural MS techniques used are native MS, ion-mobility MS (IM-MS)
and hydrogen-deuterium exchange MS (HDX-MS).
Chapter 2 combines native MS and IM-MS with cryo-EM, computational and biochemical
data to gain mechanistic insights into the assembly and ligand interactions of the HerANurA
DNA repair complex. It describes the nucleotide interactions with HerA-NurA and
their role in DNA translocation and processing. Overall, this chapter proposes a detailed
stepwise assembly mechanism of the HerA-NurA complex, leading to processing and
repair of double-stranded DNA. Chapter 3 establishes the importance of structural
dynamics in governing drug export and inhibition of the major multidrug efflux pump
AcrB. AcrB confers multidrug resistance by expelling a wide range of structurally diverse antibiotics to the outside of bacterial cells. In this work, HDX-MS together with functional
assays and computational studies were used to probe changes in the structural dynamics
of AcrB upon antibiotic and inhibitor binding. The effect of a multidrug-resistant mutation
on drug efflux is also investigated. Furthermore, the experimental workflow necessary to
study the dynamics of AcrB in its native lipid environment using HDX-MS is explored and
optimised.
Chapter 4 focuses on the Sec translocon, responsible for the transport of proteins across
the membrane. The bacterial SecYEG channel associates with the motor protein SecA to
mediate the ATP-dependent transport of pre-proteins across the membrane. HDX-MS in
combination with computational studies revealed the molecular mechanism of the
nucleotide-dependent conformational changes in SecYEG and SecA that promote the
forward transport of the pre-protein through Sec machinery.
Overall, this research highlights the power of combining structural MS together with
biochemical and computational techniques to obtain detailed mechanistic insights into
the structure, dynamics and ligand interactions of large protein complexes involved in
regulating fundamental cellular processes.
| Date of Award | 1 May 2020 |
|---|---|
| Original language | English |
| Awarding Institution |
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| Supervisor | Argyris Politis (Supervisor) & Maria Conte (Supervisor) |