Some Unanswered Questions in Membrane Science

Sol M. Gruner

Physics Dept. & CHESS

Cornell University

Before considering the ways with which x-ray techniques can help answer questions in membrane science, it is useful to first enunciate some of the “big” unanswered questions. As scientists we spend much of our time working gregariously on questions where some recent new method or insight leads us to believe that rapid progress can be made in understanding Nature. In other words, we spend much of our time huddled together under the veritable lamppost looking for coins of knowledge in the light of present understanding. This is appropriate. However, it is important to recognize that viewed from a larger perspective, the big questions in most any area of science are largely unanswered, in no small measure because it is to at all obvious how to go about attacking these questions. Still, it is important to be aware of these big questions are, since they will be the ones that preoccupy the field for many years. Below, I list a number of “big” unanswered questions about biomembranes. I choose to focus on biomembranes for two reasons. First, understanding biomembranes is one of the most important and topical research areas in biology. Second, insights on biomembranes have proven in the past to be transferable to membrane science in nonbiological areas, such as separation and chemical technology.

Why do biomembranes contain so many kinds of lipids? How does Nature decide which membranes to insert into a given kind of biomembrane, i.e., what rules are being used to make this decision? Biomembranes consist very roughly of half polar lipids and half imbedded proteins. There are many proteins and literally thousands of chemically distinct lipids in any one given type of biomembrane. Whereas the proteins are each believed to serve specific functions, such as a channel or some enzymatic function, most lipids seem to serve the more generic structural function of forming the bilayer matrix. Although some lipids serve specific messenger functions, meaning that they bind to specific binding sites in proteins, many do not appear to have specific binding sites. By various tricks of metabolism or diet, whole classes of lipids may be removed from the membrane. In response the cell will adjust the ratios of many, if not most, of the other lipids available to it. It appears that the cell is responding to the omission of the lipids by adjusting the composition of the other lipids to achieve some set of physical conditions. This process is repeatable and quantitative. Yet we do not know the rules that are being followed in making these adjustments.

Given the chemical structure of a specific lipid or mixture of lipids and a specific solvent composition, how can we predict the phase diagrams of the lyotropic system? The first step in understanding the physics and chemistry of a lipid system is to understand the phase diagram. Polar lipid/water dispersions have some of the richest phase diagrams known. Although we have learned some of the general rules that govern the topology of the diagrams, and in some cases, can predict some of the mesomorphic structures that result, we still cannot predict the detailed form of practically any lipid/water phase diagram. This illustrates the limitations of our knowledge of these important systems. The ability to predict the phase diagram would have an enormous impact on biology, as well as on many areas of science and technology.

Does Nature use biomembrane lipid composition to regulate lipid monolayer spontaneous curvature? Advances in understanding the physics of lipid/water phase diagrams has taught us in recent years that most biomembranes are composed of lipid monolayers that are under a frustrated state of elastic curvature stress. Is regulation of this parameter one of the rules that governs biomembrane lipid composition? There are enticing hints that this is the case, but this is still controversial.

If the answer to questions #3 is affirmative, is this to regulate integral membrane protein function? The realization that the lipids monolayers that make up biomembrane bilayers are under curvature elastic stress immediately suggests a specific set of mechanisms whereby this stress can couple to conformational changes of integral membrane proteins. The predicted magnitude of this coupling is on the order of several kT of energy, precisely the energy scale involved in many conformational changes.

Membrane proteins work in a qualitatively different way than aqueously soluble proteins. How, and to what extent, does Nature use distributed physical fields to modulate protein function? Integral membrane proteins differ from water-soluble proteins in that they exist in an anisotropic fluid medium. This introduces qualitatively different mechanisms of interacting with the environment. In addition to curvature elastic stress mentioned above, the liquid crystal bilayer allows imposition of huge electric fields, anisotropic chemical conditions, and elastic fields due to the bailer thickness and rigidity.

What are the structures of membrane proteins? What are the structure-function relationships unique to the membrane environment? Structural information will be needed to understand the answers to question # 5. This information is now being developed as more and more membrane protein structures are solved by x-ray crystallography. Examples include recent work by MacKinnon on voltage-gated ion channels. Another candidate is the coupling operative in the stretch-activated channels studied by Fred Sachs.

What are the properties, distributed fields, and effects on proteins of asymmetric lipid bilayers? The vast majority of work done on model lipid systems involves compositionally symmetric bilayers. Yet biomembranes always contain compositionally asymmetric bilayers. The physical properties and kinetics of asymmetric systems needs to be compared to symmetric systems of the same overall composition.

Recent work on lipid rafts has demonstrated in-plane coexistence of liquid crystalline phases. What are the phase properties and effects on biomembrane proteins and properties? Liquid-liquid phase coexistence in bilayers in now conclusively demonstrated in model membrane systems. The biological relevance is not understood.

These are just a few of many “big questions” extant in biomembrane science. X-ray methods have been among the most important structural probes used to date to understand what we presently know about biomembranes. While x-rays methods will not be the exclusive means to probe the questions posed above, each of these questions requires an understanding of molecular structure. Thus, there is no doubt that x-ray methods will continue to be used increasingly to answer the big questions of biomembranes.