Scientists from the Max Planck Institute of Biochemistry in Martinsried, Germany, and the Institute of Structural Biology in Grenoble, France, used neutron beams to expand current knowledge on structure-function relations in the ion pump bacteriorhodopsin. By using isotope-labeled membranes, areas of different dynamical behavior were detected, and a functionally essential Glycolipid was localized.
A collaboration between biochemists Heiko Patzelt and Dieter Oesterhelt from Munich's Max Planck Institute of Biochemistry and biophysicists Martin Weik, Valérie Réat and Giuseppe Zaccai from the Institute of Structural Biology in Grenoble yielded new and unusual insights into the structure-function relations in the light-driven proton pump bacteriorhodopsin. Using neutron spectroscopy and diffraction experiments, which are sensitive to isotope labels, areas of differential dynamical behavior within the protein, as well as the position of functionally important lipids surrounding it, were localized. The discoveries were recently published in Molecular Cell (1998, vol. 1, 411-419) and in the Proceedings of the National Academy of Science of the USA (1998, vol. 49, 4970-4975).
Membrane proteins play key roles in the functioning of the living cell. Apart from very few exceptions, their structure-function relations, however, still remain unclear. The light-driven proton pump bacteriorhodopsin, the photosynthetic retinal-protein in the cell walls of the salt-loving microorganism Halobacterium salinarum, can be prepared in selectively isotope-labeled forms. In addition to neutron diffraction (Journal of Molecular Biology 1989, 210, 829-847) and Nuclear Magnetic Resonance (NMR) investigations (Journal of Biomolecular NMR 1997, 10, 95-106), this now allowed the use of neutron spectroscopy to address specific dynamics which has, for the first time, been tackled in a membrane protein. The experiments were performed at the High-Flux Neutron Reactor of the Institut von Laue-Langevin in Grenoble.
Elastic incoherent neutron scattering (EINS) experiments on selectively deuterated bacteriorhodopsin, which make it possible to define amplitudes of thermal motions in the nano- to picosecond time-scale, have revealed a dynamic transition from a harmonic to a softer, inharmonic atomic fluctuation regime in the global behavior of the protein. Biological activity in proteins is correlated with this transition, suggesting that this type of flexibility is a prerequisite for function. The thermal motions of the labeled atoms in the heart of the protein, however, demonstrate stiffer behavior and, at room temperature, they have significantly smaller amplitudes of motion. Contrary to the bulk mean behavior, they appear to be shielded from solvent melting effects. These experimental results quantify the dynamical heterogeneity of bacteriorhodopsin, which meets the functional requirements of bulk flexibility, on the one hand, to allow large conformational changes in the molecule, and of a harder core, on the other, to control stereo-specific conformation changes of the central retinal.
It was shown here that protein atoms move in potential wells of different shapes and temperature characteristics, depending on their location in the structure. The EINS experiments have proven to be sufficiently sensitive to quantify this dynamical heterogeneity. The neutron approach yields the most direct and possibly unique quantitative data to test models from molecular dynamics simulations because the observable ranges of amplitude and time-scale coincide for theory and experiment. With more such data on hand, it will be possible to address important further questions: has a dynamics-function relation put evolutionary pressure on the specific dynamics of proteins, does specific dynamics have to compromise between the requirements of stability and those of function, and has specific dynamics adapted to different conditions of solvent environment, temperature or pressure?
Evidence is accumulating for the lateral organization of cell membrane lipids and transmembrane proteins in the context of sorting or intracellular signaling (Simons and Ikonen, Nature 1997, 387, 569-572). So far, however, information has been lacking on the details of protein-lipid interactions in such aggregates. Bacteriorhodopsin naturally occurs in highly ordered patches, the so-called Purple Membranes, which contain only one type of protein and a defined number of lipids. This provided a unique opportunity to study the structure of a natural membrane at sub-molecular resolution by diffraction methods.
A new method was developed to incorporate, in vivo, deuterium labels exclusively into the glycolipid sugar moieties of the Purple Membranes, and neutron diffraction experiments revealed the crystallographically ordered positions of two glycolipids per bacteriorhodopsin monomer, thus representing the first direct localization of a glycolipid with respect to a transmembrane protein in its natural membrane environment. The positions of the glycolipids strongly suggest stacking interactions between aromatic residues protruding out of the protein and the sugar moieties of the lipids, and provide direct support for the hypothesis that such interactions may play a wider role in the anchoring of membrane proteins.