Research

Membrane proteins constitute ~30% of all eukaryotic proteins (3), and are targets for over 40% of all drugs in use today (4). There is only information on very few, usually bovine membrane proteins. Until 2004 there were no structures of membrane proteins that were obtained from any other than their natural source species. These require uniquely rich sources of, for example, rhodopsin from eyes, or aquaporin 0 from fiber cells of the bovine eye lens. However the need for such rich sources is orthogonal to the need for focus onto pharmacologically important targets. The goals of the MPEC are to express such eukaryotic membrane proteins in novel ways that we have developed and optimize toward each particular class, or each particular membrane protein. Our approach therefore focuses on expression, purification and functional reconstitution of each membrane protein according to its importance for pharmacological or biological purposes.

In essentially no cases are the structures of particular membrane proteins that are drug targets yet determined. The MPEC seeks to transform the situation such that membrane proteins can be prepared and purified for use as reagents, and in the quality for crystallographic structure determination. Membrane proteins that mediate many cellular processes include transporters of molecules into and out of the cell, ATP driven pumps, receptors for neuronal signaling in the brain, channels in cardiovascular regulation, G-protein-coupled receptors that mediate vertical signaling to change metabolism of the cell very often, and receptors that mediate hormonal signaling, including both vertical signaling, and horizontal signaling by changing associations of receptors in the membrane. Despite their importance, we still know very little about the basic mechanisms by which they work. This is primarily because they are membrane proteins, where preparation, suitable incorporation into membranes, purification in structurally homogeneous forms, in functionally active states, and structure determinations have so far been very difficult.

Limited availability of pure protein prevents routine use of eukaryotic membrane proteins.

The largest limitation preventing routine biochemical use of eukaryotic membrane proteins is the limited availability of pure protein from natural or recombinant sources. To overcome this limitation, we are developing novel expression methods. This is facilitated in the development phase by coinvestigators Drs Robert Stroud, Peter Walter, David Julius, Robert Edwards, Daniel Minor, Ronald Kaback, James Van Etten, and Shimon Schuldiner, who are each experts in membrane protein biology. Assays that will be carried out at the MPEC establish stability, structural monodispersity, and oligomeric homogeneity as a key part of the purification and characterization.

Structural biologists have addressed membrane protein expression in biochemical amounts with purification, thus their final product, structures of membrane proteins are a ‘thermometer’ for the state of capabilities today. The growth in the number of ~2 to 4 Å resolution membrane protein structures now 70 in total (5 at 2 to 2.5Å resolution are from the Stroud lab) exactly parallels the exponential growth phase seen with soluble proteins, but 30 years later. Presently, there are only ~6 high-resolution structures of eukaryotic membrane proteins (1 Aqp0 from bovine eyes, from the Stroud lab), though none yet from recombinant sources (5). They are all derived from naturally rich sources. This signals the major limitation in eukaryotic membrane biochemistry. While over 60 prokaryotic membrane proteins have been over-expressed in bacteria, purified, characterized, and lead to atomic or near atomic resolution structures, essentially no attempts with eukaryotic membrane protein expression have succeeded. Our goals therefore focus on expression of membrane proteins in homogeneous and functional form.

Nevertheless, the exponential growth of the number of structures clearly signal a new capability to purify and characterize membrane proteins once they can be, or are, expressed in relatively large amounts. Prokaryotic membrane proteins, even archaeal membrane proteins have been expressed usually in E.coli or other bacterial sources, and will continue to yield biochemical, high resolution structural, and functional data. The resolutions of eukaryotic membrane protein crystals are no less than for prokaryotic or archaeal proteins: Thus there is no ‘intrinsic’ problem to eukaryotic membrane proteins per se, any more than there is for eukaryotic soluble protein crystals. However the additional requirement for functional insertion into a membrane of a heterologous organism is often a major problem. With human potassium channels huK1-4 for example, all four could be expressed in large amounts in E.coli, but in inclusion bodies, and they could never be refolded into functional channels by many procedures attempted. Thus it is the expression and insertion into a membrane, of eukaryotic membrane proteins that is the central issue that we solve here.

Expression of membrane proteins in functional form will allow determination of the molecular cell biology, and drug interactions of many therapeutically, and biologically important membrane proteins once they are expressed (1, 6). The expression outside of naturally occurring sources also allows incorporation of stabilizing mutations (7, 8), and to incorporate special atoms like selenium for structure determination (9). These procedures too can be used to produce stable, well behaved proteins for quantitative biochemistry, drug discovery, and crystallography.

Developing a means to produce large quantities of pure, functional eukaryotic membrane proteins will have a major impact on the future of medicine.

Most bacterial membrane proteins lack the rich array of regulatory domains found in eukaryotic membrane proteins and many classes of important eukaryotic proteins, such as the G-protein coupled receptors that lack bacterial homologues. Thus, it is imperative for our understanding of transmembrane signaling processes to initiate a concerted effort focused on eukaryotic membrane proteins. Developing a means to produce large quantities of pure, functional eukaryotic membrane proteins will have a major impact on efforts to understand the basic biochemistry behind transmembrane signaling and transport mechanisms that underlie processes like neurotransmission, cardiovascular regulation, and hormonal signaling, as well as the development of new drugs to treat dysfunctions in these systems.

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