Dynamics of different functional parts of bacteriorhodopsin: H-2H labeling and neutron scattering.
We show that dynamics of specific amino acids within a protein can be characterized by neutron spectroscopy and hydrogen-deuterium labeling, and we present data on the motions of a selected set of groups within bacteriorhodopsin (BR), the retinal-based proton pump in the purple membrane of halophilic Archaea. Elastic incoherent neutron scattering experiments allow the definition of motions in the nano- to picosecond time scale and have revealed a dynamical transition from a harmonic to a softer, anharmonic atomic fluctuation regime in the global behavior of proteins. Biological activity in proteins is correlated with this transition, suggesting that flexibility is required for function. Elastic incoherent neutron scattering is dominated by H atom scattering, and to study the dynamics of a selected part of BR, fully deuterated purple membrane with BR containing H-retinal, H-tryptophan, and H-methionine was prepared biosynthetically in Halobacterium salinarum. These amino acids cluster in the functional center of the protein. In contrast to the protein globally, the thermal motions of the labeled atoms were found to be shielded from solvent melting effects at 260 K. Above this temperature, the labeled groups appear as more rigid than the rest of the protein, with a significantly smaller mean square amplitude of motion. These experimental results quantify the dynamical heterogeneity of BR (which meets the functional requirements of global flexibility), on the one hand, to allow large conformational changes in the molecule and of a more rigid region in the protein, on the other, to control stereo-specific selection of retinal conformations.
Réat
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Institut de Biologie Structurale Commissariat à l'Energie Atomique-Centre National de la Recherche Scientifique, 41 Avenue des Martyrs, F-38027 Grenoble Cedex 1, France.
Patzelt
H
H
Ferrand
M
M
Pfister
C
C
Oesterhelt
D
D
Zaccai
G
G
D000596:Amino Acids / Q000737:chemistry; D001436:Bacteriorhodopsins / Q000737:chemistry*; D002462:Cell Membrane / Q000648:ultrastructure; D003903:Deuterium; D006217:Halobacterium / Q000648:ultrastructure; D007501:Iron / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D008958:Models, Molecular; D009038:Motion; D009502:Neutrons; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation
D000596:Amino Acids; D008565:Membrane Proteins; D001436:Bacteriorhodopsins; D003903:Deuterium; D007501:Iron