Determining structural dynamics of membrane proteins in their native environment: focus on bacterial antibiotic resistance

Cellular health is determined by the structure, movement, and interplay of its biomacromolecules. Being able to interrogate the behaviour of biomacromolecules within a native cellular context would enable us to gain an enhanced understanding of how these molecules dictate a cells behaviour and function. Proteins are an essential class of biomacromolecule which perform a wide range of cellular processes such as enzyme catalysis, cell signalling and scaffolding, and DNA replication. They consist of a linear chain of amino acids, defined as a polypeptide, their sequence being determined by the genetic sequence which encodes them. An important subset of proteins is integral membrane proteins which reside within cellular membranes and account for about 30% of cellular proteins. Cellular membranes are dynamic structures consisting mostly of protein and lipid which act to compartmentalise the cell, providing barriers to the external environments of the cell and its organelles. Integral membrane proteins are defined by their content of hydrophobic polypeptide stretches which enable parts of their structure to be embedded within, or associated with, the cellular membrane. They are responsible for a variety of dynamic cellular processes, such as sensation, cellular regulation, and cell-to-cell adhesion. A membrane protein's functional capability and their level of expression will largely decide the ionic composition, and therefore the metabolic levels of a given cell type, making them essential for all life, as well as key drug targets.

My main aim is to determine structural dynamic information of membrane proteins directly within their native cellular membrane environment, including within live cells. It is important to understand the structural dynamics of proteins, as their fluctuations frequently represent motions and states that are critical for protein function. To do this I will develop general strategies which enable membrane protein structure and dynamics to be deciphered within complex environments by advanced structural mass spectrometry methods. Structural mass spectrometry uses high-resolution mass information on polypeptides and their peptide building blocks to infer on the structural properties of a protein molecule - their shape, interactions, and movements. Using techniques such as hydrogen/deuterium exchange mass spectrometry (which measures the extent and rate of exchange of protein backbone amide hydrogens for deuterium), both global and local information on protein interactions, ligand binding, and structural dynamics can be delivered. Here, I propose the development of chemical biology and advanced mass spectrometry strategies for membrane protein structural investigation within different native membrane environments.

One key area in which integral membrane proteins are important is in the development of antimicrobial resistance. Combating antimicrobial resistance is a key societal challenge which, if not addressed, has the potential to become a global health crisis. In bacterial cell lines, the development of multiple drug resistance to structurally unrelated chemicals have been correlated to the function of multidrug efflux membrane protein transporters, which expel a broad range of toxic substances and result in reduced inhibitory effects of antibiotics. My research will focus on developing the aforementioned methods in the context of multidrug efflux membrane protein systems which are known to play major roles in bacterial antibiotic resistance. This will enable an unprecedented insight into the structure, dynamics, and function of these systems, particularly on the impact of drug and lipid interactions, and clinically relevant mutations. More generally, the ability to achieve structural insight into biomacromolecules within cells would be a huge step forward in our understanding of how they shape the function of healthy and diseased cells.

Impact on the third and public sectors:

Important to this research proposal is the development of techniques and protocols that will allow new structural insights into the mechanisms of multidrug efflux membrane protein transporters, which play major roles in bacterial antibiotic resistance. Thus, understanding and developing techniques to probe these systems will have long term economic and societal impacts in improving quality of life and health. Especially in the generation of novel tools for in vivo membrane proteins structural interrogation, which will contribute greatly to the expertise and health of multi-disciplinary areas used to investigate complex processes.

By participating in public outreach activities (facilitated by the Widening Participation scheme at KCL) the proposal will allow me to raise public awareness of the potential benefits of enhancing bioanalytical science and its potential benefit on health and pharmaceutical research. For example, taking part in Discover Science Days which are open to KS4 and 5 school students and their parents and offer a unique opportunity to find out more about studying physical sciences at undergraduate level. Through this participation I expect to inspire, encourage and enthuse young school students to pursue further education and careers in bioanalytical disciplines for health, particularly in areas such as drug discovery and antimicrobial resistance.

Impact on the commercial sector:

Intellectual property arising from the research is expected. By identifying and protecting any arising intellectual property, commercial opportunities arising from the work and approaches can be capitalized upon. If new intellectual property is generated pursuits to license the technology with pharmaceutical or bioanalytical service companies or even develop a 'spin-out' company associated with King's College London will be made, contributing to economic development. R&D investment and/or collaboration will be pursued with companies interested in the development of the technology, such as Waters (mass spectrometry), GlaxoSmithKline and UCB (biopharmaceutics). I have previous experience with licensing products for bioanalytical science - with a licensed patent for the use of mass spectrometry in drug discovery (licensed by OMass Technologies in 2016) - and working previously with the pharmaceutical company GlaxoSmithKline.

Laboratory members will be trained (3-8 members over the course of the fellowship) in novel and emerging structural mass spectrometry methodologies, as well as in molecular biology, cell biology, structural biology, and membrane protein biochemical investigation. These techniques are important for drug discovery in non-academic professions, therefore, by training and developing highly skilled people in these disciplines the proposal will have both a societal and economic impact. I have demonstrated my ability to train graduate students through my experience in both student supervision and lecturing. I have continuously co-supervised PhD/Master students and solely supervised my own summer student in 2016, both involved allocating projects and laboratory training, with my summer student's work contributing to a recent publication in Angewandte Chemie. I have also attended the EMBO laboratory management course for group leaders which strengthened my laboratory and people management skills, teaching in areas such as laboratory leadership, support, organisation, and effective communication.

Timescales:

Impact on the third and public sectors and intellectual property generation will come immediately after research publication, dissemination, and outreach; predicted to be within 2 - 7 years of the Fellowship. Economic and societal impacts will likely come with advancement of the methodologies and creation of higher throughput bioanalytical tools that enable many drug targets to be screened and assessed; this is predicted to be anywhere between 5 - 10 years.