seience/technology
Lights, Camera, What Action? New evidence reopens old dispute on bacteriorhodopsin isomerization
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Hsion and photosynthesis are made possible through a complex cascade of events that is not fully understood, but is the source of heated argument. Recently published research [Proc. Natl. Acad. Sci. USA, 94, 7937 (1997)] has prompted another flare-up in the controversy about what happens when light hits a photosynthetic pigment, bacteriorhodopsin, that is similar to the human visual pigment rhodopsin. Bacteriorhodopsin, a pigment found in patches in the membranes of Halobacterium, is composed of the retinylidene chromophore retinal linked through a Schiff base to the protein bacterioopsin. When light hits these patches, bacteriorhodopsin shifts protons out of the bacterial cell, creating a proton gradient that serves as a source of energy. The retinal in bacteriorhodopsin undergoes isomerization when it interacts with light, shifting from the all trans form to a 13-cis form. Applied science professor Aaron Lewis, physical chemistry professor Michael Ottolenghi and graduate student Edward Khachatryan at Hebrew University of Jerusalem, working with organic chemistry professor Mordechai Sheves and graduate student Itay Rousso at Weizmann Institute
of Science in Rehovot, Israel, have come up with a technique to directly probe conformational alterations in proteins. They used it to study bacteriorhodopsin's interaction with light. "The universally accepted mechanism for the photochemistry of retinylidene proteins is the cis-trans photoisomerization model, in which the primary action of light is to photoisomerize the retinylidene chromophore," Lewis says. But he and his colleagues believe results from their new atomic force sensing technique, which "monitors protein conformational changes that have been previously silent in conventional spectroscopy," call the accepted mechanism into question. The device rests the tip of a glass-fiber cantilever, developed by Lewis and his colleagues, on the surface of a preparation of membrane fragments containing bacteriorhodopsin. Pulses of laser light beamed at the membranes are absorbed by the bacteriorhodopsin molecules, which then change shape. The subsequent movement of the cantilever is monitored by reflecting a different laser beam off a metallized portion of the cantilever onto a position-sensitive detector. The technique has a time resolu-
tion—the lag between protein motion and the response of the equipment—of just a couple of microseconds, "more than three orders of magnitude faster than any previous attempt at ultrafast atomic force sensing using conventional silicon cantilevers," Lewis says. The Israeli workers used their technique to study both native bacteriorhodopsin and an artificial pigment analog in which the trans-cis isomerization typical of bacteriorhodopsin was blocked. To their surprise, this so-called locked version also underwent a protein conformational change when exposed to light, similar to that of the native system. The researchers conclude "that such conformational changes do not involve trans-cis isomerization." They suggest instead that light absorption initiates a charge redistribution in the retinal chromophore, pushing it into a higher energy state. This in turn, they propose, induces conformational changes in the surrounding protein matrix as the structure seeks to return to a lower energy state. The researchers say further work will be needed to clarify the nature of the protein conformational changes and their role in bacteriorhodopsin's biological function. They believe bacteriorhodopsin responds to light with charge redistribution—rather than the isomerization of the chromophore—and that this is what triggers the protein conformational change. Lewis, who proposed this possibility almost 20 years ago (C&EN, March 13, 1978, page 22), says the latest results represent the first experimental proof that protein conformational alteration can occur without retinal trans-cis isomerization.
Bacteriorhodopsin's response to light remains controversial Classical model
13-c/s-Retinal trans-Retinal Protein in dark state
Altered functional
Protein in dark state
protein
Altered protein
Lewis model
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mophore returns to its original trans ground state. Warshel doesn't think such a short-lived change is sufficient to bring about a lasting conformational change in the protein. He draws an analogy with a door held closed by a strong spring, which represents the protein in its initial equilibrium, low-energy state. "If you have a system in equilibrium, and you try to move it to another equilibrium, you can't do it just by giving it a very fast push and then reLewis uses an atomic force sensing technique to monitor treating," as in Lewis' conformational alterations in photosynthesis proteins. model, Warshel says. "If But, Arieh Warshel, professor of chem- you hit the door very fast and then pull istry and biochemistry at the University of your fist back, there is no known mechaSouthern California, Los Angeles, believes nism that will keep the door open." that Lewis' results can be explained with Warshel thinks instead that the chroWarshel's own model, which he has also mophore must be stabilized in its new been advocating for nearly two decades. state long enough to bring about a correWarshel does agree with Lewis that a sponding change in the protein, or, in photon of light initiates a charge redistribu- the terms of the analogy, to hold the tion in the chromophore. But he says that in door open. Warshel proposes that the the Lewis model this ends when the chro- bonds in the chromophore undergo a
Alligator secretions yield diterpenoids Movie scenes of sharp-toothed jaws locking on a hapless victim fuel the reputation of alligators as ferocious carnivores. Most people would not seek to be within biting distance of these animals, unless it's their business to study them. One person who studies alligators is Paul J. Weldon, a zoologist at the Conservation & Research Center of the Smithsonian Institution, Front Royal, Va., shown here collecting secretions from an American alligator at the St. Augustine Alligator Farm, St. Augustine, Fla. The secretions probably contain pheromones for attracting mates and marking nests. Recently, his alligator research, in collaboration with chemists, has found a diterpene hydrocarbon called cembrene-A and a related ketone in the oily secretions of adult male Chinese alligators (Alligator sinensis). Cembrene-A is usually associated with higher forms of plants, although it has been found in corals and insects. The recent work represents thefirstisolation of cembrene-A from a vertebrate [/. Nat Prod, 60, 828 (1997)]. It is also the first study to isolate and characterize the ketone, which differs from cembrene-A in that the C-ll/C-12 bond is saturated and position 10 bears a carbonyl group.
The compounds were isolated from yellow, citrus-smelling secretions of glands near the cloaca, called the paracloacal glands. Only the Chinese alligator produces the diterpenoids. The American species (A. mississipiensis) does not. Weldon says the Chinese alligator is an endangered species now found only
change upon interaction with light, with the single bonds shortening and the double bonds lengthening. (Warshel notes, incidentally, that such "bond alternation" is common in other related molecules.) This reshaped chromophore, with its new charge distribution, is stable for long enough to force a corresponding change in the protein's conformation and to keep it there. The chromophore is then able to shift from trans to cis "in a subsequent stage without too much effort,* he says. On the other hand, without bond alternation, "you would have to work too hard to move from trans to cis once the light energy is gone." Lewis responds that his 1978 model shows that the light-altered protein stabilizes the charge redistribution induced by light in the chromophore. And he notes that his team's technique detects protein coordinate changes that have gone undetected by other techniques that focus on the chromophore. Looking beyond the bacteriorhodopsin controversy, Lewis believes the results obtained from the sensing method have implications for understanding the behavior of other chromoproteins, including visual pigments. Sophie Wilkinson in a few provinces in China. They are also being raised in seminatural enclosures at the Rockefeller Wildlife Refuge, Grand Chenier, La., where they are being bred through a program of New York City's Bronx Zoo. "Such a breeding program—and similar ones at other zoos on other species—have made exotic animals available for natural products studies," says Weldon. Maureen Rouhi
Cembrene-A
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