SCIENCE & TECHNOLOGY
OPEN UNDER R PRESSURE Biophysical study showss how membranes open Is swell the floodgates when cells AMANDA YARNELL, C&EN WASHING!TON
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cell membrane separating them from their rapidly changing environments. Excessive moisture can cause a bacterium to swell and eventually burst, so it relies on members of a class of membrane proteins known as mechanosensitive channels that act like emergency pressure-relief valves: When the internal osmotic pressure gets too high, mechanosensitive channels open wide, allowing water, ions, and small molecules to rush out to relieve the pressure. Until recently scientists have been able to glean few molecular details about how pressure on the membrane is transduced into protein motion. But now, Eduardo Perozo, a professor of molecular physiology and biological physics at the University of Virginia Health Sciences Center, and colleagues are using electron paramagnetic resonance (EPR) plus a few other clever tricks to figure out how these channel proteins sense and react to changes in pressure on the surrounding membrane. Perozo reported his lab's recent efforts in this area at the 47th annual meeting of the Biophysical Society, which took place in San Antonio, Texas, early last month. His talk was one offour selected from more than 150 nominations to be presented in a special symposium showcasing cuttingedge biophysical research. W h e n water builds up inside a thinskinned bacterium, high osmotic pressure deforms the membrane by changing the tension parallel to the membrane. This membrane deformation then causes changes in mechanosensitive channels' structure, opening them up. It's this deformation that remains mysterious. Researchers have suggested two possible explanations for how pressure-induced changes in the lipid membrane might trigger opening of these channels. When pressure impinges on the membrane, it "thins" locally Part of the channel that was submerged in the membrane could then be exposed to water and the resulting hydrophobic mismatch could cause the channel to open. Or the pressure difference between the inside and the outside of the cell could asymmetrically curve the membrane, exerting lateral pressure on 34
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CHANNEL CHANGING Perozo's lab has created nearly 70 versions of MscL, each modified with a spin label at a different spot (yellow dots) on the protein. EPR analysis of these showed that the transmembrane helices (shown in red and blue) tilt to open the channel like the iris of a lens. the channel and thereby forcing it open. Because nature has built mechanosensitive channels to tend to stay closed—and therefore leakproof—it's been hard to test these two proposals. Plus, channels opened by high osmotic pressures stay open for only a few nanoseconds, making it difficult to probe the open channel's structure. Perozo and his collaborator, Boris Martinac of the University of Western Australia, have tried to shed light on this question by cutting out the pressure problem and forcing the mechanosensitive channel called MscL to
open by chemically manipulating its membrane environment. They "thin'' the membrane surrounding the channels by substituting lipids with shorter acyl chains. But, Perozo says, "Thinning is not enough to open the channel completely" HOWEVER, using lipids with different geometries is. By using a cone-shaped lipid called lysophosphatidylcholine in the membranes, Perozo fools the channel into thinking that pressure has been applied. "This simple trick has allowed us to study the open channel for the first time," he says. Douglas C. Rees, a crystallographer at California Institute of Technology whose lab determined the X-ray crystal structure of the closed mechanosensitive channel [Science, 282,2220 (1998)}, calls Perozo's lipid trick "a beautiful example" ofhow understanding the basic principles of membrane biophysics can be used to manipulate the lipid content of the membrane in order to stabilize the channel's normally disfavored open state. This breakthrough, Rees notes, allowed Perozo to overcome a critical problem common to the study of any protein exhibiting multiple conformations: Less stable conformations often don't stick around long enough for biophysical studies. Perozo and Martinac are probing the open channel—as well as the closed channel and a partially open intermediate—by applying elegant EPR site-directed spinlabeling methods pioneered by Perozo's postdoc mentor, biochemist Wayne L. Hubbell of the University of California, Los Angeles. Hubbell's technique involves creating a specific "sticky" spot on the protein by mutating a single amino acid residue to cysteine. He then attaches a nitroxide spin label probe via a disulfide bond to this sticky spot. By analyzing the spin-labeled protein's EPR spectrum, he can gather information about the label's local environment in the protein. With enough of these spin-labeled proteins, he can glean global information about the protein's structure
By measuring the spectral changes in spin labels, researchers have been able to show that the mechanosensitive channel opens like the iris of a lens. and—more important—he can follow changes in the protein's structure during function in real time. Hubbell has used the technique to investigate the dynamics of HTTP://WWW.CEN-ONLINE.ORG
the retinal proteins rhodopsin and bacteriorhodopsin, as well as a variety of other proteins. Perozo has also used this method to nail down how the helices that make up the channel rearrange to create an open pore thought to reach a diameter as large as 30 A. "This is perhaps one of the largest conformational changes known in membrane proteins," he tells C&EN. Rees's 1998 crystal structure of the closed channel had led his and a number of other labs to surmise that the channel prob-
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Nitroxide spin labels are tethered to the protein at engineered cysteine residues via disulfide linkages. ably opened by repositioning the tightly packed helices like the staves of a barrel. Recently however, chemical cross-linking and computational efforts by biologist Sergei Sukharev of the University of Maryland, College Park, have called this simple barrel stave model into question [Nature, 409,720 (2001)}. SukhareVs experiments have led him to propose an alternative mechanism in which the MscL channel opens like the iris of a lens. This model requires that the innermost helices tilt with respect to the membrane surface, flattening the channel and opening a wide pore. Perozo's group's spin-labeling studies neatly clear up the controversy Technician D. Marien Cortes and postdoc Pornthep Sompornpisut have created nearly 70 versions of MscL, each modified with a spin label at a different spot on the protein. By measuring the spectral changes in spin labels on different MscLs, as well as the accessibility of each spin label to chemical reagents located in water or in the membrane, Perozo and his collaborators have been able to show that, as Sukharev has suggested, MscL opens like the iris of a lens. But so far, Perozo has only looked at the structural differences between the closed channel, a partially closed intermediate, and the open channel. Hubbell points out, however, that EPR site-directed spin labeling can be used to watch the conformational changes that take place during channel gating in real time. Perozo tells C&EN that he and his coworkers are now trying to film that very movie. • HTTP://WWW.CEN-ONLINE.ORG
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