Exploring magnetically aligned bilayers as a novel tool for membrane protein crystallisation

Membrane-embedded proteins (or membrane proteins) are amongst the most influential for the survival, correct behaviour and function of cells. They form the means by which cells interact with their environments, by which they import nutrients and expel potentially poisonous molecules, and communicate with each other. In humans, membrane proteins comprise almost a third of all known proteins, and over half of the currently available drugs act on membrane proteins. Thus, studying the roles of membrane proteins and how they function is a pursuit shared by both academic research scientists and the pharmaceutical industry alike.
A powerful means for studying the role and function of proteins is visualising their three-dimensional shape with enough detail to distinguish individual chemical groups and atoms. Such analysis allows us to understand the purpose of each protein component as if we were observing a machine, and to envision ways of assisting or disrupting its mechanism that can then be translated into drugs and therapies for diseases. X-ray crystallography is the premier method by which we visualise proteins at this level of detail; however, this method requires the formation of highly ordered crystals where protein molecules pack against each other in a predictable and regular manner. Due to the fact that membrane proteins need to be extracted from their natural membrane environment in order to be crystallised, they are often damaged and therefore they do not easily form crystals. It is for this reason that, despite their enormous importance in living organisms, membrane proteins make up a very small proportion, less than 2%, of proteins for which the detailed shape is known. Thus, developing novel tools that induce membrane proteins to form crystals could tremendously expand our detailed understanding of cellular mechanisms.
Traditionally, membrane proteins were isolated and handled in the presence of soap-like detergent molecules; however, such detergents make the proteins less likely to function correctly or to form crystals. For this reason, researchers have been developing advanced methods that provide a more membrane-like environment for the proteins during crystallisation, e.g. by the addition of lipids that are similar to those in the cell membrane. When membrane proteins do crystallise through these methods the crystals often consist of spontaneously formed stacks of lipid bilayers, an arrangement that vaguely resembles the situation the protein would encounter in the membrane of a living cell. In this proposal we aim to develop a new method that assists the regular packing of membrane proteins in such stacked lipid bilayers, and thereby increases the probability that they form crystals. To do so we will utilise strong superconducting magnets, which are known to impose order in membranes by forcing their orientation to follow the direction of the magnetic field. We hypothesise that in this way membrane-embedded proteins will also be forced toward a particular direction, and this spatial restriction may induce them to pack more readily into crystals. Should this magnetic alignment crystallisation ('MAX') approach prove successful, we aim to further develop the use of magnets in the crystallisation of membrane proteins into a tool widely available in the academic community and industry.

Membrane proteins comprise 'high value' targets for structural analysis as understanding their shape and function at the amino acid level is often linked to developing new small molecule therapeutics. Indeed, membrane proteins constitute 59% of approved drug targets for a variety of conditions including cancer, hypertension and clinical depression. Yet, to date, only 722 unique membrane protein structures are known, comprising less than 2% of all structurally characterised proteins. A major obstacle in the structural analysis of membrane proteins by X-ray crystallography is the low success rate of crystal formation by these proteins. Furthermore, even when membrane protein crystals do form they are often smaller and produce weaker X-ray diffraction data compared to crystals of soluble proteins. Thus, there is strong interest in developing advanced methods to counteract these crystallisation difficulties.
Recent successes in membrane protein crystallisation relied on bilayer systems that spontaneously form small regions of organised membrane lamellae, which support the formation of 3D crystals composed of stacked 2D protein sheets. Here, we propose to assist this type of protein organisation and packing by externally inducing spatial order in the lamellar phase of lipid-water mixtures. To that end we will utilise strong magnetic fields that can force the alignment of membrane lamellae along an axis parallel to the applied field. Membrane proteins embedded in these bilayers will, thus, have restricted orientational freedom, which may accelerate crystal growth and make protein packing more regular leading to improvements in X-ray diffraction potential. We will initially assess the suitability of this magnetic alignment crystallisation (MAX) approach using a well characterised membrane protein (SERCA); in addition, we also plan to gather information on the general utility of the MAX method by exploring the crystallisation of uncharacterised membrane proteins.

We propose here the development of a novel methodology that has the potential to facilitate the structure determination process for membrane proteins. By extending and improving our understanding of membrane protein structure we envision that our research will lead to societal (1) and economic (2) impacts. Membrane proteins are directly relevant to medical conditions and to human health, and they constitute the most common class of drug targets; therefore they are directly relevant to pharmaceutical R&D leading to investment and exploitable commercial products. Thus, we envision two groups, beyond academics, who would benefit from the proposed research:
1) Patients and medical professionals responsible for their treatment will gain from improved understanding of how membrane proteins function. Dysfunction in membrane proteins is associated with a wide range of conditions, including cystic fibrosis, Alzheimer's disease, cystinuria and renal diabetes insipidus amongst others. Often, our understanding of the origins of these conditions is limited as our knowledge of the canonical and mutant structures of the relevant membrane proteins is incomplete. Should this structural information be available, medical practitioners, for example, would be able to judge the likely impact of mutations identified in early embryos or choose among therapeutic courses aimed at ameliorating protein dysfunction. Our research aims to assist the structure determination process and thereby precisely fill in the missing information on the mechanisms of membrane proteins. Thus, our endeavours may enable fundamental advancements in bioscience that will then drive better health outcomes for the society as a whole. 'Biosciences for health' is a strategic priority area for BBSRC.

2) Of the 646 separate human proteins that are related to the mechanism of action for drugs over half (379) are membrane bound. Well known examples include drugs for the detection of thyroid cancer that utilise the thyroid hormone GPCR, antihypertensive compounds that target calcium channels, antidepressants targeting the serotonin transporter and psychotropic drugs bound by dopamine transporters. Drugs modulate the normal behaviour of proteins by tightly associating with them; however, to discover initial compound hits and develop them into effective and commercially viable drugs knowledge of the protein structure in its free and drug-bound states is essential. Visualising the sites on proteins where drugs bind allows us to design compounds that fit these sites almost as key to a lock. Thus, research towards new methods for determining membrane protein structures at high resolution can directly assist the drug discovery process. Pharmaceutical R&D makes a substantial contribution to the national economy both through sales of drug products and through the creation of highly skilled jobs, and support for such R&D is a key priority for BBSRC.