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

Lead Research Organisation: King's College London
Department Name: Chemistry

Abstract

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.

Planned Impact

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.

Publications

10 25 50
 
Description Member of the Foundation Future Leaders Programme
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
Impact The aim of the programme is to develop links and understanding across Government, Parliament, industry and the research community, by establishing a cohort of 30 people, consisting of 8 government civil servants, two parliamentary civil servants, 10 researchers and 10 people from industry. There will be a series of meetings and visits for this cohort - which I have a strong lead in designing - and a conference at the end of the year. In addition, members of the cohort will be invited to all of the main discussion debates of the Foundation for Science and Technology. It is understood that Foundation Future Leaders will take part in discussions on topics which bridge the space between research, government, Parliament and industry.
 
Description PostDoc Tutor for KCL Chemistry
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Influenced training of practitioners or researchers
Impact In my capacity as Postdoc Tutor I birthed the inaugural PostDoc Chemistry Community at King's College London. The PostDoc Chemistry Community (PostDoc ChemComm) represents the research and career development of postdocs, contracted researchers and postdoc fellowship holders within the Department of Chemistry at King's College London. We are a platform open to every researcher who has wondered how to improve their research and career, how to get better impact or who simply wants to share their experiences and concerns within the community. We disseminate possible funding and careers opportunities for our post-doc's and organise post-doc focused seminars and social events. In my role as Postdoc tutor I passionately foster a thriving research community and facilitate a productive and rewarding environment for early career researchers. I feed into departmental meetings about post-doc issues and support the running of PostDoc ChemComm in partnership with their post-doc representatives. I also feed into faculty meetings about post-doc issues through continued attendance and cooperation with the NMS Postdoctoral Research Staff Committee, who in turn will attempt to implement KCL wide changes.
URL https://www.kcl.ac.uk/chemistry/postgraduate
 
Description BBSRC iCASE LIDo studentship in partnership with Waters Corporation 
Organisation Waters Corporation
Department Waters Corporation
Country United Kingdom 
Sector Private 
PI Contribution The student will develop structural mass spectrometry and protein engineering approaches to decipher native membrane protein structural behaviour. The project will involve advancement and training on cutting-edge H/D eXchange mass spectrometry (HDX-MS) for HDX structural analysis of membrane protein dynamics. With an emphasis being placed on understanding the role of drugs and the lipid metabolism on protein function. Importantly, the student will perform molecular biology and biochemical investigations alongside technology development to fully understand membrane protein behaviour and function.
Collaborator Contribution This project is performed in partnership with Waters Corporation which will include a 3-month placement at the Waters Mass Spectrometry Headquarters facility at Wilmslow, UK. During their placement, Waters Corporation will provide education and training on how a large scientific company is organised and run. This will include meeting members from a variety of departments and disciplines, both formally and informally, to understand their business strategy, hierarchy, and how teams work together.
Impact None as of yet - studentship application date closes on 23rd March 2020.
Start Year 2020
 
Description Dr Attilio Vargiu 
Organisation University of Cagliari
Country Italy 
Sector Academic/University 
PI Contribution Structural mass spectrometry and biophysical analysis on the multidrug efflux transporter AcrB in the presence of antibiotics and inhibitors. Studying the wildtype protein and a clinically relevant multidrug resistance (MDR) mutation.
Collaborator Contribution Molecular dynamics (MD) simulation analysis on the multidrug efflux transporter AcrB in the presence of antibiotics and inhibitors. Studying the wildtype protein and a clinically relevant multidrug resistance (MDR) mutation.
Impact Manuscript in preparation.
Start Year 2019
 
Description Prof Charles Deber 
Organisation Hospital for Sick Children, Toronto
Country Canada 
Sector Hospitals 
PI Contribution Native mass spectrometry analysis of peptide-based inhibitors (designed in the Deber lab) impact on multidrug efflux pump AcrB structure, to aid in tackling antibiotic and multidrug resistance in bacteria.
Collaborator Contribution Deisn and synthesis of peptide-based inhibitors against multidrug efflux pump AcrB function, to aid in tackling antibiotic and multidrug resistance in bacteria.
Impact Manuscript currently under review at Journal of Biological Chemistry in which I am co-corresponding (senior) author.
Start Year 2019
 
Description Prof Laura Piddock 
Organisation University of Birmingham
Country United Kingdom 
Sector Academic/University 
PI Contribution Structural mass spectrometry and biophysical analysis on the multidrug efflux transporter AcrB in the presence of antibiotics and inhibitors. Studying the wildtype protein and a clinically relevant multidrug resistance (MDR) mutation.
Collaborator Contribution Microbiological analysis of the multidrug efflux transporter AcrB in the presence of antibiotics and inhibitors. Studying the wildtype protein and a clinically relevant multidrug resistance (MDR) mutation.
Impact Manuscript in preparation.
Start Year 2019
 
Description Prof Mark Howarth 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution Developing structural mass spectrometry approaches for the determination of native membrane protein structural biology information. Using these developments, alongside molecular biology and biochemical investigation, to decipher the mechanisms of multidrug efflux transporters which play major roles in bacterial antibiotic resistance.
Collaborator Contribution Providing expertise, materials and experiments on the use of covalent and non-covalent binding protein partners (designed in the Howarth lab) to facilitate structural mass spectrometry investigations of proteins in native environments.
Impact None as of yet - collaboration just begun.
Start Year 2020
 
Description Appointed Member of the BBSRC Pool of Experts 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Supporters
Results and Impact Pool of Experts members typically serve on BBSRC Research Committees, leading on the assessment of their responsive mode applications.
Year(s) Of Engagement Activity 2020
 
Description Co-organised a internationally recognized Biochemical Society conference on "Structural Mass Spectrometry of Membrane Proteins" 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This two-day meeting will focus on new advancements and technological developments in mass spectrometry for studying the structure and function of membrane proteins. It will bring together MS practitioners and structural biologists from academia and industry and will include sessions across many aspects of structural mass spectrometry of membrane proteins.

The conference programme will focus on the ever-growing developments and applications of structural mass spectrometry of membrane proteins and their complexes, as well as new advancements and technological developments in mass spectrometry.

Topics for discussion will include the latest developments in instrumentation, intact membrane protein complexes, data analysis, software development and a range of applications to membrane proteins, such as lipid and drug interactions, dynamics of transporters and channels, membrane protein folding, and the study of large macromolecular complexes (e.g. ATPases).

The inaugural "Structural Mass Spectrometry of Membrane Proteins" meeting successfully brought together academic and industrial scientists who are working at the forefront of the technology.

We saw presentations on how structural mass spectrometry can decipher membrane protein structure and function, either as a standalone tool or as a powerful integrative technique alongside other structural biology methods. We had examples of how it has been applied to medically important membrane protein systems such as, GPCRs, ATPases, and those involved in antibiotic resistance and cancer. A highlight was a dedicated session on the development of structural mass spectrometry for protein analysis within live cells - seeing the expansion of the technique to living cells (and even live worms!) acting to inspire where this field can go.

A key success was the dissemination and sharing of ideas which will enable the incredible work of our (relatively) small field to be applied and developed further - with the hope that non-specialist users will increasingly utilize these techniques to answer their most important membrane biology questions. From this meeting we could see that the field is expanding and its reach into structural biology is being increasingly welcomed. We, therefore, see this meeting as just the beginning.
Year(s) Of Engagement Activity 2019
URL https://biochemistry.org/events/structural-mass-spectrometry-of-membrane-proteins/
 
Description Invited to speak at the 11th BBSRC Fellows' Conference in Glasgow, UK 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Welcome Message from Melanie Welham, BBSRC Executive Chair:

I am very pleased to welcome you to the 11th BBSRC Fellows' Conference. We have held these conferences biennially since 1999, and I hope you will find this event to be as stimulating and as beneficial as past conferences have been. I am proud to be Executive Chair of BBSRC as we hold this latest conference. As many of you will know as a result of my own BBSRC Fellowship that I held earlier in my career I attended previous conferen
Year(s) Of Engagement Activity 2019
 
Description Invited to speak at the Membrane Protein Folding, Gordon Conference in Boston, USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The 2019 GRC on Membrane Protein Folding will be the 3rd biannual GRC of this new series. While this series is rooted in the membrane biophysics and biochemistry communities, a hallmark of the 2019 program is the participation of a distinguished subset of invited speakers who are experts on the cell biology of membrane protein folding. An exciting frontier exists at the interface between the cell biology and biophysics of membrane protein folding, and this meeting will stimulate exploration and collaboration in this domain. Other topics to be covered in the 2019 program include the presence of native disorder in membrane proteins, the role of membrane protein folding in retinitis pigmentosa, how folding and receptor signaling can be dynamically linked, the specificity of lipid-protein interactions in folding, how membrane proteins and respond to changes in temperature, integration of nascent membrane proteins into cell membranes, and engineering of membrane proteins. The program is designed to encourage participation by young scientists in that there will be some 20 short (15 minute) talks selected from the abstracts, complementing the usual poster sessions.

I gave a 25 minute talk on "Advanced Mass Spectrometry to Probe Membrane Proteins in Native Membranes" which was well received and sparked many questions and discussions on my research afterwards.
Year(s) Of Engagement Activity 2019
URL https://www.grc.org/membrane-protein-folding-conference/2019/
 
Description Performed the Chemistry Christmas Lecture as part of the Faculty of Natural & Mathematical Sciences Christmas Lecture Series 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact The Christmas Lectures give secondary school pupils from the ages of 15-18 an opportunity to explore a variety of Christmas themed topics in science, technology, engineering and mathematics (STEM) through lectures and demonstrations from university teaching staff. There will also be an opportunity to have a tour of our Strand Campus and meet current students and academics from the faculty.

The talks were:

Once upon a time, there was a photon who helped Santa to freeze time by Dr Amelle Zair
How to win your Christmas family board game by Professor Igor Wigman
Father Christmas' naughty or nice detector by Dr Emma Robinson
Santa's little helper: A robot that can travel inside your body by Dr Hongbin Liu
The gift wrap of life by Dr Eamonn Reading
All I want for Christmas is...a robot that can do my homework by Dr Helen Yannakoudakis
Year(s) Of Engagement Activity 2019
URL https://www.kcl.ac.uk/events/christmas-lectures-2019
 
Description Synthesized the Reading Group website 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Dedicated website to the outputs and news of the Reading group has been set up to aid in research dissemination, group recruitment, and collaborative activity.
Year(s) Of Engagement Activity 2019
URL https://www.ereadinglab.com/