Deciphering the conformational mechanisms of nascent membrane protein folding

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.

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.

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.