Deciphering the conformational mechanisms of nascent membrane protein folding

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

Abstract

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 reside within a cellular membrane and are responsible for a variety of dynamic 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.
Membrane proteins must be folded 'correctly' to become functional. The key aim of the research proposal is to understand how nascent membrane proteins fold. The chemical principles of membrane protein folding during their production and in the context of a cellular membrane are poorly understood. Investigating membrane proteins is a difficult task due to the intractable and hydrophobic nature of membrane proteins, compared to their soluble protein counter parts; with additional protein machinery being required for their cellular localisation and folding within the membrane, in comparison. To understand the processes involved would enhance our understanding on how the cell obtains 'correctly' folded and functional membrane proteins, and postulate on how 'incorrectly' misfolded and aggregated membrane proteins are formed - a toxic process for a cell. Consequently, development of robust assays that reflect the cellular processes will enable the influence of co-factors, mutations and drugs on the process of membrane protein folding. Moreover, there is a key aim to develop methodologies to gain structural insight into membrane protein events using advanced chemical tools and techniques, such as hydrogen-deuterium exchange mass spectrometry.
The research is proposed to take place at the King's College London, Department of Chemistry, within the laboratory of Professor Paula Booth. The research will involve producing systems capable of both membrane protein production, insertion and folding in vitro and developing methodologies for the structural assessment of these processes with strong focus on using hydrogen deuterium exchange mass spectrometry.

Technical Summary

Integral membrane proteins reside and function within cellular membranes. Membrane proteins must be folded 'correctly' to become functional. During folding integral membrane proteins are highly prone to aggregation and insolubility in aqueous solutions (such as in the cytoplasm of the cell) due to protein regions possessing high degrees of hydrophobicity. Additional cellular machinery is therefore required for membrane protein insertion and folding into its final membrane environment. Membrane protein folding can occur co- or post-translationally. In prokaryotes this occurs primarily with the utilization of the Sec translocon and translocase insertion protein machinery, although co-translational insertion using other insertases is also prevalent, as well as spontaneous insertion. Their final membrane organization and topology is determined by the polypeptide interaction(s) with the translocase machinery, intramolecular interactions within the protein, and intermolecular interactions between the protein and its final membrane environment. There is a wealth of biochemical research on the translation process and its machinery, however, the investigation of - and more importantly the development of methods and techniques capable of monitoring - the structural dynamics of co-translational nascent membrane protein folding has been distinctly lacking. The research goal is to use complementary biochemical techniques alongside hydrogen deuterium mass spectrometry - which reports on the solvent accessibility of polypeptides - to gain unprecedented insight into the mechanisms of this fundamental cellular process. It is proposed that the membrane protein, the disulphide bond forming protein B (DsbB), would be an ideal candidate for investigating the mechanisms of nascent membrane protein folding. DsbB is a component of the disulphide bond formation pathway that enables the formation of disulphide bonds of periplasmic proteins in bacteria, and a target for antivirulence drugs.

Planned Impact

Understanding fundamental cellular processes inherently brings with it wide interest and academic impact from a broad range of disciplines. A couple of high profile examples being translational tuning of nascent soluble protein folding and chaperone-assisted folding; both of these areas either form a component, or are akin, to the overarching premise of this research proposal: to probe and understand membrane protein folding in the context of its translation and membrane insertion. More focused benefits will be in the area of general protein research, contributing to fundamental scientific areas, such as the 'protein folding problem', and benefitting academic communities such as the Protein Society.
Important to this research proposal is the development of techniques and protocols that will allow structural insight into the mechanisms of nascent membrane protein folding and will have immediate academic impact upon publication. The development of in vitro membrane protein production systems with proteoliposomal systems demonstrates advancements that will benefit the wider chemical biology community; through the generation of responsive and manipulative in vitro systems that can, importantly, inform on cellular processes. Moreover, developing new methodologies which permit coupling between cell-free systems with hydrogen deuterium mass spectrometry equipment will contribute greatly to the expertise and health of multi-disciplinary areas (short term benefits especially within mass spectrometry and 'omics' communities) used to investigate complex processes.
By using the bacterial membrane protein, the disulphide bond forming protein B (DsbB), the influence and delivery of co-factors on membrane protein folding can also be investigated - as it requires the lipophilic ubiquinone co-factor for its function - a deprived area of research. In addition, DsbB is involved in disulphide bond formation of periplasmic proteins and because these disulphide bonds confer stability on many of these proteins, numerous which are bacterial virulence factors, DsbB has been become a drug target for inhibiting bacterial virulence. Thus, understanding and developing techniques to probe this system could have long term economic and societal impacts in improving quality of life and health.
The availability of high resolution structural information for DsbB enables the structurally dynamic results gained from hydrogen deuterium mass spectrometry to be collated, thus providing a detailed picture of its translation and insertion. Collating data in this way will help to accomplish goals similar to that expressed by the Membrane Protein Structural Dynamic Consortium (MPSDC): to use state of the art spectroscopic and spectrometric methods in combination with high-resolution structural information to gain structural information on membrane protein folding and dynamics.
The timescales of these impacts will vary with the academic impact coming immediately after publication of the research predicted to be within 2 - 4 years if the Fellowship is obtained. Economic and societal impacts will likely come when the advancement of methodologies and creation of higher throughput bioanalytical tools so that many drug targets can be screened and assessed; this is predicted to be anywhere between 5 - 10 years.

Publications

10 25 50
 
Description Successfully folded membrane proteins underlie a plethora of cellular processes. The folding of proteins from an amino acid sequence to their functional state is essential to life, but little is known about the co-translational process, where nascent proteins fold during synthesis by ribosomes. I have advanced our knowledge on the nascent folding of the most understudied protein class; those that reside within cell membranes. By applying numerous biochemical (including radiolabelling experiments) and biophysical techniques coupled with cell-free transcription translation systems I was able to show that a lipid bilayer devoid of any translocon components is sufficient for successful co-translational folding of two different a-helical membrane proteins, DsbB and GlpG. The ability to form a properly folded, functional state from an amino acid sequence produced in vitro without key in vivo machinery indicates that the correct fold of a-helical membrane proteins is an equilibrium structure, independent of the folding pathway. Additionally, I was able to show that the lipid membrane (a membrane proteins natural environment) is able to influence, and control, the success of this process. Moreover, surface-enhanced infrared absorption spectroscopy (SEIRAS) was used to investigate membrane protein folding, where structure formation of the two proteins was monitored during their in vitro co-translational folding. This work was published in Scientific Reports (2017). Note: the SEIRAS technique was used because it is superior at monitoring both structure formation and insertion during co-translational folding of membrane proteins (and is a very new, exciting technique).

Membrane proteins are difficult to study using mass spectrometry, and many other biophysical techniques, due to their inherent hydrophobicity. To perform hydrogen-deuterium exchange mass spectrometry (HDX-MS) on ribosome nascent complexes (RNC) of membrane proteins therefore requires a series of analytical advances - one being the ability to study membrane proteins within native and complex cellular membrane environments. I have currently developed a general novel HDX-MS approach which can inform on the conformational dynamics of membrane proteins within native nanodiscs (SMALPs). The SMALP technology enables membrane proteins to be captured within a nanodisc formed by the SMA polymer surrounding a native lipid bilayer - this enables membrane proteins to be studied within the context of their native environment. I have succeeded in HDX-MS analysis of GlpG in SMALPs. This work was published in the high impact journal Angewandte Chemie Int Ed (2017) and follow up work with LILBID mass spectrometry in Chemical Communications and Nature Communications (2018). The next step is to be able to generate stable RNCs of GlpG which have the potential to be interrogated by HDX-MS. I have been able to generate stable GlpG RNCs in vivo and purify them to homogeneity within native bilayers (using native nanodisc polymer technology). To do this I used a SecM cDNA approach with different affinity tags (e.g. hexahistidine and Avi tags) cloned at the nascent chains N-terminus. The next step is to be able to interrogate these RNCs by HDX-MS. I have devised a method for this and am currently testing and optimizing protocols. I am beginning to be able to understand the capabilities of HDX-MS for studying membrane proteins and RNCs and current experiments suggest it has a bright future. Some of these results are currently being written up for publication.
Exploitation Route My findings have been published within reputable journals and presented at both invited seminars and international conferences. This will hopefully enable researchers to take my developed insights and methods and apply them to their research.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Other

 
Description Collaboration with Dr Argyris Politis group, KCL 
Organisation King's College London
Country United Kingdom 
Sector Academic/University 
PI Contribution I contributed to the design of the biochemistry and structural mass spectrometry experiments and analysis.
Collaborator Contribution They designed and performed all experiments and wrote the manuscript.
Impact This work was published: Ahdash Z., Lau A. M., Byrne R. T., Lammens K., Stuetzer A., Urlaub H., Booth P. J., Reading E., Hopfner K-P., Politis A., Mechanistic insight into the assembly of the HerA-NurA helicase-nuclease DNA end resection complex, Nucleic Acid Res., 20, 12025-38. (2017)
Start Year 2016
 
Description Collaboration with Dr. Zoe Hall, University of Cambridge 
Organisation University of Cambridge
Department Department of Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution We designed the project, performed all experiments except the lipidomics, performed the HDX-MS experiments, and wrote the manuscript with contributions from all authors.
Collaborator Contribution Dr. Zoe Hall at the University of Cambridge performed the lipidomics for this work.
Impact This work was published in: Reading E.†, Hall Z., Martens C., Haghighi T., Findlay H., Ahdash Z., Politis A., Booth P. J., Interrogating membrane protein conformational dynamics within native lipid compositions, Angew. Chem. Int. Ed., 49, 15654-57. (2017) †Corresponding authorship
Start Year 2016
 
Description Collaboration with Freie Universität Berlin 
Organisation Free University of Berlin
Country Germany 
Sector Academic/University 
PI Contribution We designed the research, performed all experiments except for SEIRAS experiments, analysed SEIRAS data, and wrote the manuscript with contributions from all authors.
Collaborator Contribution They designed the research, performed SEIRAS experiments, and analysed SEIRAS data.
Impact This work was published: Harris N. J.†, Reading E.†, Ataka K., Grzegorzewski L., Charalambous K., Liu X., Schlesinger R., Heberle J., Booth P. J., Structure formation during translocon-unassisted co-translational membrane protein folding, Sci. Rep., 7, 8021. (2017) †Co-lead authorship
Start Year 2016
 
Description Dr. Nina Morgner 
Organisation Goethe University Frankfurt
Country Germany 
Sector Academic/University 
PI Contribution We designed the project together. I performed experiments for the encapsulation of folded membrane proteins within native lipid bilayers for structural interrogation. I performed biochemical, biophysical, and biochemical characterization of these complexes, including native mass spectrometry.
Collaborator Contribution The Morgner lab performed laser induced liquid bead ion desorption mass spectrometry on these membrane protein complexes to understand their structural and non-covalent interactions. They wrote the paper (with contributions from other authors).
Impact This work was published in: Hellwig N., Peetz O., Ahdash Z., Tascón I., Booth P. J., Mikusevic V., Diskowski M., Politis A., Hellmich Y., Hänelt I., Reading E., and Morgner N., Native mass spectrometry goes more native: investigation of membrane protein complexes directly from SMALPs, Chem. Comm., 54, 13702-05. (2018)
Start Year 2017
 
Description Annual guest lecturer on interrogating membrane proteins to undergraduates 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact Around 30 undergraduate students attend this 1 hour guest lecture each year to learn about how to study membrane proteins as well as the membrane protein research I undertake in the Department of Chemistry at King's College London. The lecture has been well received and students have shown an interest in (my) research not on their syllabus.
Year(s) Of Engagement Activity 2016,2017
 
Description Post-doctoral tutor for Department of Chemistry 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact I recently (in 2018) became the PostDoc Tutor for the department of Chemistry at King's College London and instated the departments first PostDoc Chemistry community. 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 the PostDoc Chemistry community in partnership with post-doc representatives. I also feed into faculty meetings through cooperation with the NMS Postdoctoral Research Staff Committee, who in turn implement University wide changes.

A particular focus of this group is to help postdoctoral researchers achieve fellowships, such as the BBSRC Discovery fellowship (formerly the BBSRC Future Leader Fellowship). Using my fellowship experiences and disseminating these, as well as my research, to these groups has enabled the fostering of postdoc's for future fellowship applications. We have had an increase in these types of applications and interest in applying for postdoctoral fellowship money since the inception of this group.

A few impacts include:

Development of a postdoc forum: Casual meeting of post-docs and final year PhD students to discuss what you want from a PostDoc Community and how we can help facilitate this. This will be a platform open to every single researcher who wondered how to improve their research and career, how to get better impact or who simply want to share their experiences and concerns within the community. We will also provide some information on relevant fellowships and KCL career training available in the next few months.

Development of a postdoc community website: Website to support the postdoc community and provide regular dissemination on the postdoc community and career development.

Organisation of career talks e.g.: I organised Dr Liz Elvidge (Imperial College London) to present at KCL. She is a well-renowned postdoc career expert. This talk was opened up to the entire NMS, so that the department postdoc's would also have an opportunity to meet other postdocs from across the NMS. 35 postdocs were ticketed to attend and the event was well received with many debate and questions sparked after the talk. The postdoc community report an interest in more career events like this. Once such future event we are organizing is:

NMS Careers event (8th May 2019): The event will focus on non-academic careers, and ways to transition from an academic role.
Year(s) Of Engagement Activity 2018,2019
URL https://www.kcl.ac.uk/nms/depts/chemistry/postdoc-community/post-doc-chemcomm
 
Description Presentation at the 62nd Annual Meeting of the Biophysical Society. February 17-21, 2018, San Francisco, California 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Performed 'Interrogating membrane protein conformational dynamics within native lipid bilayers with hydrogen-deuterium exchange mass spectrometry' presentation at the Biophysical Society 62nd Annual Meeting, in San Francisco, California, USA, 17-21st February, 2018. Each year, the Biophysical Society Annual Meeting brings together more than 6,000 researchers working in the multidisciplinary fields representing biophysics. With more than 3,600 poster presentations, over 200 exhibits, and more than 20 symposia, the BPS Annual Meeting is the largest meeting of biophysicists in the world. My presentation was well attended and instigated interesting and useful discussions during the conference and after.
Year(s) Of Engagement Activity 2018
 
Description Question and answer session on the achievements of BBSRC fellowship(s) at the Roslin Institute, Edinburgh 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact I was invited to perform a question and answer session on the achievement of my current BBSRC Future Leader Fellowship (FLF). This was on a panel with two other BBSRC FLF fellows, two BBSRC David Phillips Fellows and a senior member of the BBSRC research grants team.
Year(s) Of Engagement Activity 2018