Unfolding Pathways of Individual Bacteriorhodopsins Oesterhelt, F.; Oesterhelt, D.; Pfeiffer, M. ...
Science (American Association for the Advancement of Science),
04/2000, Letnik:
288, Številka:
5463
Journal Article
Recenzirano
Atomic force microscopy and single-molecule force spectroscopy were combined to image and manipulate purple membrane patches from Halobacterium salinarum. Individual bacteriorhodopsin molecules were ...first localized and then extracted from the membrane; the remaining vacancies were imaged again. Anchoring forces between 100 and 200 piconewtons for the different helices were found. Upon extraction,the helices were found to unfold. The force spectra revealed the individuality of the unfolding pathways. Helices G and F as well as helices E and D always unfolded pairwise, whereas helices B and C occasionally unfolded one after the other. Experiments with cleaved loops revealed the origin of the individuality: stabilization of helix B by neighboring helices.
The dynamical coupling between proteins and their hydration water is important for the understanding of macromolecular function in a cellular context. In the case of membrane proteins, the ...environment is heterogeneous, composed of lipids and hydration water, and the dynamical coupling might be more complex than in the case of the extensively studied soluble proteins. Here, we examine the dynamical coupling between a biological membrane, the purple membrane (PM), and its hydration water by a combination of elastic incoherent neutron scattering, specific deuteration, and molecular dynamics simulations. Examining completely deuterated PM, hydrated in H₂O, allowed the direct experimental exploration of water dynamics. The study of natural abundance PM in D₂O focused on membrane dynamics. The temperature-dependence of atomic mean-square displacements shows inflections at 120 K and 260 K for the membrane and at 200 K and 260 K for the hydration water. Because transition temperatures are different for PM and hydration water, we conclude that ps-ns hydration water dynamics are not directly coupled to membrane motions on the same time scale at temperatures <260 K. Molecular-dynamics simulations of hydrated PM in the temperature range from 100 to 296 K revealed an onset of hydration-water translational diffusion at almost equal to200 K, but no transition in the PM at the same temperature. Our results suggest that, in contrast to soluble proteins, the dynamics of the membrane protein is not controlled by that of hydration water at temperatures <260 K. Lipid dynamics may have a stronger impact on membrane protein dynamics than hydration water.
An integrated picture of hydration shell dynamics and of its coupling to functional macromolecular motions is proposed from studies on a soluble protein, on a membrane protein in its natural lipid ...environment, and on the intracellular environment in bacteria and red blood cells. Water dynamics in multimolar salt solutions was also examined, in the context of the very slow water component previously discovered in the cytoplasm of extreme halophilic archaea. The data were obtained from neutron scattering by using deuterium labelling to focus on the dynamics of different parts of the complex systems examined.
Heterogenous nucleation on small molecule crystals causes a monoclinic crystal form of bacteriorhodopsin (BR) in which trimers of this membrane protein pack differently than in native purple ...membranes. Analysis of single crystals by nano-electrospray ionization-mass spectrometry demonstrated a preservation of the purple membrane lipid composition in these BR crystals. The 2.9- angstrom x-ray structure shows a lipid-mediated stabilization of BR trimers where the glycolipid S-TGA-1 binds into the central compartment of BR trimers. The BR trimer/lipid complex provides an example of local membrane thinning as the lipid head-group boundary of the central lipid patch is shifted by 5 angstrom toward the membrane center. Nonbiased electron density maps reveal structural differences to previously reported BR structures, especially for the cytosolic EF loop and the proton exit pathway. The terminal proton release complex now comprises an E194-E204 dyad as a diffuse proton buffer.
We report the sequence of the
Halobacterium salinarum strain R1 chromosome and its four megaplasmids. Our set of protein-coding genes is supported by extensive proteomic and sequence homology data. ...The structures of the plasmids, which show three large-scale duplications (adding up to 100 kb), were unequivocally confirmed by cosmid analysis. The chromosome of strain R1 is completely colinear and virtually identical to that of strain NRC-1. Correlation of the plasmid sequences revealed 210 kb of sequence that occurs only in strain R1. The remaining 350 kb shows virtual sequence identity in the two strains. Nevertheless, the number and overall structure of the plasmids are largely incompatible. Also, 20% of the protein sequences differ despite the near identity at the DNA sequence level. Finally, we report genome-wide mobility data for insertion sequences from which we conclude that strains R1 and NRC-1 originate from the same natural isolate. This exemplifies evolution in the laboratory.
Lag1p and Lac1p are two homologous transmembrane proteins of the endoplasmic reticulum in Saccharomyces cerevisiae. Homologous genes have been found in a wide variety of eukaryotes. In yeast, both ...genes, LAC1 and LAG1, are required for efficient endoplasmic reticulum-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins. In this study, we show that lag1 Delta lac1 Delta cells have reduced sphingolipid levels due to a block of the fumonisin B1-sensitive and acyl-CoA-dependent ceramide synthase reaction. The sphingolipid synthesis defect in lag1 Delta lac1 Delta cells can be partially corrected by overexpression of YPC1 or YDC1, encoding ceramidases that have been reported to have acyl-CoA-independent ceramide synthesis activity. Quadruple mutant cells (lag1 Delta lac1 Delta ypc1 Delta ydc1 Delta) do not make any sphingolipids, but are still viable probably because they produce novel lipids. Moreover, lag1 Delta lac1 Delta cells are resistant to aureobasidin A, an inhibitor of the inositolphosphorylceramide synthase, suggesting that aureobasidin A may be toxic because it leads to increased ceramide levels. Based on these data, LAG1 and LAC1 are the first genes to be identified that are required for the fumonisin B1-sensitive and acyl-CoA-dependent ceramide synthase reaction.
Components of biological macromolecules, complexes and membranes are animated by motions occurring over a wide range of time and length scales, the synergy of which is at the basis of biological ...activity. Understanding biological function thus requires a detailed analysis of the underlying dynamical heterogeneity. Neutron scattering, using specific isotope labeling, and molecular dynamics simulations were combined in order to study the dynamics of specific amino acid types in bacteriorhodopsin within the purple membrane (PM) of Halobacterium salinarum. Motions of leucine, isoleucine and tyrosine residues on the pico- to nanosecond time scale were examined separately as a function of temperature from 20 to 300 K. The dynamics of the three residue types displayed different temperature dependence: isoleucine residues have larger displacements compared to the global PM above 120 K; leucine residues have displacements similar to that of PM in the entire temperature range studied; and tyrosine residues have displacements smaller than that of the average membrane in an intermediate temperature range. Experimental features were mostly well reproduced by molecular dynamics simulations performed at five temperatures, which allowed the dynamical characterisation of the amino acids under study as a function of local environment. The resulting dynamical map of bacteriorhodopsin revealed that movements of a specific residue are determined by both its environment and its residue type.
Structural changes of peptides containing the azobenzene dye 4-aminomethyl-phenylazobenzoic acid (AMPB) are studied with ultrafast spectroscopy. AMPB peptides are a new class of molecules where the ...photoisomerizable dye azobenzene is linked to the peptide moiety via a flexible methylene spacer. The ultrafast reactions in the femtosecond to nanosecond time domain are investigated for the optical switch AMPB, a linear and cyclic octapeptide, and a bicyclic octapeptide containing an additional disulfide bridge. These molecules with increasing conformational constraints are studied for the
cis to
trans and the
trans to
cis photoreactions. For the
cis to
trans reaction the isomerization of the chromophore occurs fast in the 1-ps range, whereas it is slower (10-ps range) in the
trans to
cis reaction. In all peptides the structural changes of the chromophore lead to modifications in the peptide structure in the 10-ps–1-ns time range. The results indicate that the chromophore AMPB acts simultaneously as a fast molecular switch and as a sensor for initial conformational dynamics in the peptide. Experiments in the mid-infrared range where the structural changes of the peptide backbone are directly observed demonstrate that the essential part of the structural dynamics in the bicyclic AMPB peptide occurs faster than 10
ns.
Studies have shown that trans-cis isomerization of retinal is the primary photoreaction in the photocycle of the light-driven proton pump bacteriorhodopsin (BR) from Halobacterium salinarum, as well ...as in the photocycle of the chloride pump halorhodopsin (HR). The transmembrane proteins HR and BR show extensive structural similarities, but differ in the electrostatic surroundings of the retinal chromophore near the protonated Schiff base. Point mutation of BR of the negatively charged aspartate D85 to a threonine T (D85T) in combination with variation of the pH value and anion concentration is used to study the ultrafast photoisomerization of BR and HR for well-defined electrostatic surroundings of the retinal chromophore. Variations of the pH value and salt concentration allow a switch in the isomerization dynamics of the BR mutant D85T between BR-like and HR-like behaviors. At low salt concentrations or a high pH value (pH 8), the mutant D85T shows a biexponential initial reaction similar to that of HR. The combination of high salt concentration and a low pH value (pH 6) leads to a subpopulation of 25% of the mutant D85T whose stationary and dynamic absorption properties are similar to those of native BR. In this sample, the combination of low pH and high salt concentration reestablishes the electrostatic surroundings originally present in native BR, but only a minor fraction of the D85T molecules have the charge located exactly at the position required for the BR-like fast isomerization reaction. The results suggest that the electrostatics in the native BR protein is optimized by evolution. The accurate location of the fixed charge at the aspartate D85 near the Schiff base in BR is essential for the high efficiency of the primary reaction.
Structural changes are central to the mechanism of light‐driven proton transport by bacteriorhodopsin, a seven‐helix membrane protein. The main intermediate formed upon light absorption is M, which ...occurs between the proton release and uptake steps of the photocycle. To investigate the structure of the M intermediate, we have carried out electron diffraction studies with two‐dimensional crystals of wild‐type bacteriorhodopsin and the Asp96‐‐>Gly mutant. The M intermediate was trapped by rapidly freezing the crystals in liquid ethane following illumination with a xenon flash lamp at 5 and 25 degrees C. Here, we present 3.5 A resolution Fourier projection maps of the differences between the M intermediate and the ground state of bacteriorhodopsin. The most prominent structural changes are observed in the vicinity of helices F and G and are localized to the cytoplasmic half of the membrane.
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