Sideline Viewing of the BR Membrane Protein

For this article, Leslie Pray interviewed Hartmut "Hudel" Luecke, professor of molecular biology and biochemistry, University of California, Irvine. Data from the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. H. Luecke B. Schobert, H.T. Richter, J.P. Cartailler, J.K. Lanyi, "Structure of bacteriorhodopsin at 1.55 Å resolution," Journal of Molecular Biology, 291[4]:899-911, 1999. (Cited in 105 papers) Courtesy of Harmut LueckeA bacteriorhodopsin crystal before (left) and after (right) increasing illumination In the mid-1970s, electron microscopy provided the first visualization of the membrane protein bacteriorhodopsin (BR), a photon-driven ion pump found in the salt-marsh dwelling bacterium Halobacterium salinarum. Since then, BR has become one of the most-studied and best-understood membrane proteins. Still, its underlying molecular mechanism remains a major, unsolved problem of membrane bioenergetics. In this Hot Paper, scientists captured BR images at a high enough resolution that let them finally "start to piece together a detailed mechanism for how light drives the pump," says Hartmut "Hudel" Luecke, professor of molecular biology and biochemistry, University of California, Irvine. Luecke and colleagues used x-ray diffraction to observe the structure of bacteriorhodopsin at 1.55 Å resolution, "the highest resolution x-ray structure of any membrane protein," Luecke says. Membrane proteins are embedded in the cell membrane bilayer, serving as gatekeepers, enabling signals, solutes, and other important cellular currency to traverse the otherwise insoluble bilayer. Some act like channels or sensors. Others, like the BR ion pump, either passively or actively transport nutrients and other biochemicals. Scientists know the structure of about 30 membrane proteins, explains Luecke, even though one-quarter of most genomes code for them. "There are many more protein membranes than people like to think." These proteins are very unstable and don't readily crystallize, making it difficult to collect the kind of x-ray data commonly used to study protein structure. But developments in high-resolution x-ray crystallography have given scientists a sophisticated new tool, cubic lipid phase (CLP) crystallization,1 which serves as a "whole new way to coerce membrane proteins to crystallize," Luecke says. He and his colleagues used this technique to achieve the results described in this Hot Paper. They added the BR proteins to a CLP matrix where, within a few days, crystals grow large enough to be seen under a light microscope. "You can almost see them with the naked eye," says Luecke, "they are purple and very beautiful." CLP reveals the pump's atomic structure in more detail than before. As Luecke puts it, "Now we have a structure where we know the position of every atom. We can now see all the internal non-hydrogen atoms and all the bound [water molecules], and we can see the lipids and how they interact with the protein.... We see structure and a surprising amount of shape-complementarity between the surface of the membrane-embedded protein and the membrane lipids." The paper's results are based on a BR image in its ground state. In a follow-up study,2 the researchers used the same x-ray diffraction technique to "capture a snapshot of the pump in an intermediate state half-way around the pump cycle," according to Luecke. Together, these studies will allow the researchers to "start piecing together a detailed mechanism for how light drives the pump," Luecke says. "We can start deducing how light energy is converted into the electrochemical energy that in turn is used to generate ATP." BR uses light to generate an electrochemical gradient that is converted into chemical energy by another membrane protein, ATP synthase. But membrane bioenergetic researchers disagree on how the pump operates. Does it transport protons (H+) from the cytoplasmic to the extracellular side of the cell, or does it transport hydroxide ions (OH-) in the other direction?3 While the jury is still out, Luecke comments that "a better understanding of the pump's structure will help us understand what kind of pump it is." He and his colleagues continue to study other intermediary steps in the BR pump cycle, as well as structures and functions of other membrane proteins that use light as an energy source. Leslie Pray can be contacted at lpray@nasw.org. References 1. E.M. Landau and J.P. Rosenbusch, "Lipidic cubic phases: a novel concept for the crystallization of membrane proteins," Proceedings of the National Academy of Science, USA, 93:14532-5, 1996. 2. H. Luecke et al., "Structural changes in bacteriorhodopsin during ion transport at 2 angstrom resolution," Science, 286:255-60, 1999. 3. H. Luecke, "Atomic resolution structures of bacteriorhodopsin photocycle intermediates: The role of discrete water molecules in the function of this light-driven ion pump," Biochimica et Biophysica Acta, 1460:133-56, 2000.

Sideline Viewing of the BR Membrane Protein

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