Interrogating the folding and function of membrane proteins by mass spectrometry

A membrane protein is a protein that is attached to, or associated with, the membrane of a cell. The membrane is the cell's impermeable surface through which many ions, nutrients, hormones, drugs and proteins enter and exit the cell, and membrane proteins are responsible for transportation across the membrane and cell signalling, the latter of which governs the actions of the cell.

Membrane proteins are important medically. Examples of maladies in which membrane proteins are implicated include: cancer, which can involve over-production of membrane proteins and errors in cell signalling pathways; heart disease, where the regulation of ions across the cell membrane may be failing; diabetes, which has been linked with membrane protein mutations; and depression, where there is an inbalance of the levels of specific molecules in cells. Due to their association with such diseases, and their convenient location at the cell surface where they are accessible to small molecule drugs, membrane proteins account for >50 % of all known drug targets. They are also important targets for anti-bacterial agents.

Despite membrane proteins representing ~30% of all proteins, details of their characteristics and mode of action are scarce as they are notoriously difficult species to handle and analyse because of their hydrophobicity and insolubility. Compared with water-soluble proteins, few membrane proteins have been characterised structurally: only ~309 out of a total of >65,000 protein structures archived in the Protein Data Bank are of membrane proteins. Our understanding of many diseases, and our subsequent ability to treat these diseases and improve our quality of life, is reliant on a better knowledge of the structure of these proteins, the ways in which they interact with ions and small molecules, and their mode of function.

Our aim is to understand the way in which membrane proteins function by developing analytical methods with which we can monitor structural changes taking place during a folding or functional event. We will use electrospray ionisation-mass spectrometry (ESI-MS) coupled to ion mobility spectrometry, a combined technique which produces information about mass, stoichiometry, shape and stability of individual protein species present within complex mixtures in a single, rapid experiment. It is ideal for monitoring the way a protein changes its 3D shape and for monitoring reactions between proteins and other species, where the starting materials, reaction intermediates and products can be analysed simultaneously. We have pioneered this technique to provide insights into water-soluble protein functioning events in real-time and now wish to exploit its use for membrane proteins.

Prior to ESI-MS analysis we will use chemical labelling techniques to probe the 3D structures of membrane proteins at different times during a functioning event. Such methods modify the protein which results in a change of mass which we can detect. We will then use ESI-MS to analyse the individual species within the reaction and identify exactly which regions of the protein are exposed and available to react (the labelled areas), and those regions which are shielded within its 3D structure or by interactions with other species. By labelling and analysing at different times during the reaction we will produce a series of "snapshots" of the event in real-time. Together the snapshots will provide a complete picture revealing insights into the functional properties of membrane proteins.

To carry out the chemical labelling and ESI-MS analysis we will purchase laser equipment to couple to our existing mass spectrometers and then develop robust protocols that we can apply to a wide range of membrane proteins. For this development work, we have chosen two different proteins, each an example from one of the two major classes of membrane proteins, the all alpha-helical and beta-barrel, which we have considerable experience of handling.

The insolubility of membrane proteins renders their characterisation by X-ray crystallography or NMR difficult, if not impossible. We propose to combine recent innovations in electrospray ionisation-mass spectrometry (ESI-MS) with chemical labelling methods to provide insights into the dynamical properties of membrane proteins at the molecular level.

Coupling ESI-MS to ion mobility spectrometry (ESI-IMS-MS) permits information about mass, stoichiometry, shape and stability of individual species in complex mixtures to be obtained in a single experiment. We have pioneered this technique to provide insights into water-soluble proteins, monitoring folding and assembly events in real-time. Here we wish to realise the full capability of ESI-(IMS)-MS for studies of membrane proteins.

Prior to mass spectrometric analysis we will use two complementary chemical labelling techniques (photo-oxidation, hydrogen-deuterium exchange) to probe the 3D structures of membrane proteins during folding or function events. Each method modifies the protein with a resulting change in mass, from which we can determine the regions of the protein that are exposed and which are shielded within its 3D structure. "Snapshots" of the events will be analysed in real-time to provide a complete sequence of events in molecular detail. For photo-oxidation methods, we will purchase and set-up a KrF excimer laser on-line to the mass spectrometer and develop protocols for real-time analyses with high temporal resolution.

To develop methods which can be applied to a wide range of membrane proteins in the future, we have chosen two proteins as examples of the all alpha-helical and beta-barrel classes of membrane proteins. We have shown that we can analyse such proteins directly from detergent micelles by ESI-IMS-MS but, as an alternative method of protein handling under more native-like conditions, we will prepare Nanodiscs packed with protein to investigate direct analysis from lipid bilayers.

Scientific beneficiaries:
Membrane proteins, which account for ~30% of all proteins, have critical roles in many cellular processes. Despite their biomedical importance, relatively little is known about the structure and function of these species compared to the wealth of information available for globular proteins. Determining the mode in which membrane proteins function is a challenging task but will expose a whole new field of science if successful. Our project addresses the key issue of interrogating the structural dynamics of membrane proteins in real-time and will generate a wealth of new, significant information concerning the folding and function of membrane proteins, in addition to protocols which we will make widely available for future research. The results will be of interest to a broad spectrum of life-scientists, pharmacists and medical researchers in academia and industry. We have requested funding to organise a 2-day international workshop in Leeds which will focus on new biophysical methodologies developed for membrane proteins at which we, and others worldwide, will present our findings.

Industrial beneficiaries:
Our long-term collaboration with Micromass/Waters UK Ltd. has resulted in four BBSRC/CASE PhD studentships in the past decade, and Micromass/Waters have committed to provide practical and technical expertise to help us achieve our method development plans in this project, confirming their continued interest in applying mass spectrometry to solve biological problems. We have close contact with several pharmaceutical companies (AZ, GSK, Medimmune) who currently sponsor CASE students. If successful, our developments would open up a new area of protein research which currently cannot be exploited and the pharmaceutical industry would benefit hugely which, in the longer term, would lead to new and improved medicines (see letter of support from AZ). Understanding transport of hydantoins by Mhp1 is a project of importance in industrial production of amino acids by Ajinomoto Inc. initiated by PJFH. The University of Leeds has just invested £500K in a "Pharmaceutical and Biopharmaceutical Sector Hub" to extend the engagement with industry initiatives established by the Astbury Centre for Structural Molecular Biology (AEA is the Astbury Centre Industry Liaison Officer). We will use the Hub to promote our research and establish suitable industrial collaborators if/when appropriate.

Delivering highly skilled people:
The proposed project will train a PDRF in a wide range of cutting-edge technologies including state-of-the-art mass spectrometry, membrane protein handling and analysis, and a range of other biophysical techniques. This will well-equip the PDRF for a future successful career, whether in academia or industry. Highly skilled post-graduate and post-doctoral fellows from all three applicants have gone on to post-doctoral fellowships and lectureships in other universities, and positions in industry within the UK and overseas. At Leeds, we actively promote research-led teaching for both undergraduate and postgraduate students. Hence progress made in the membrane protein field would enhance many aspects of the teaching delivered and contribute to the students' learning experience. In terms of our own research, once the methods have been developed we intend to exploit them to study other membrane proteins of biological and medical interest. For this, we would anticipate significant further pharmaceutical sponsorship in addition to opportunities to apply for further grant funding from research councils and charities.

Wider impacts for the general public:
Improved knowledge of protein function will lead to improved drug developments and thence to an enhanced quality of life. Several membrane proteins have been identified as targets for pharmaceutical intervention in prevalent diseases including depression and diabetes, as well as new targets for the generation of anti-bacterial agents.