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Bioengineered 2012The properties of bacteriorhodopsin (BR) can be manipulated by genetic engineering. Therefore, by the methods of gene engineering, Asp85 was replaced individually by two...
The properties of bacteriorhodopsin (BR) can be manipulated by genetic engineering. Therefore, by the methods of gene engineering, Asp85 was replaced individually by two other amino acids (D85V, D85S). The resulting recombinant proteins were assembled into soybean vesicles retinylated to form functional BR-like nano-particles. Proton translocation was almost completely abrogated by the mutant D85S, while the D85V mutant was partially active in pumping protons. Compared with wild type, maximum absorption of the mutants, D85V and D85S, were 563 and 609 nm, which illustrated 5 nm reductions (blue shift) and 41 nm increases (red shift), respectively. Since proton transport activity and spectroscopic activities of the mutants are different, a wide variety of membrane bioreactors (MBr) have been developed. Modified proteins can be utilized to produce unique photo/Electro-chromic materials and tools.
Topics: Aspartic Acid; Bacteriorhodopsins; Electrochemical Techniques; Escherichia coli; Genetic Engineering; Ion Transport; Kinetics; Light; Mutagenesis, Site-Directed; Mutation; Nanoparticles; Photochemical Processes; Protons; Recombinant Proteins; Serine; Glycine max; Spectrum Analysis; Valine
PubMed: 22976247
DOI: 10.4161/bioe.21048 -
Journal of the American Chemical Society Mar 2018Despite much attention, the path of the highly consequential primary proton transfer in the light-driven ion pump bacteriorhodopsin (bR) remains mysterious. Here we use...
Despite much attention, the path of the highly consequential primary proton transfer in the light-driven ion pump bacteriorhodopsin (bR) remains mysterious. Here we use DNP-enhanced magic angle spinning (MAS) NMR to study critical elements of the active site just before the Schiff base (SB) deprotonates (in the L intermediate), immediately after the SB has deprotonated and Asp85 has become protonated (in the M intermediate), and just after the SB has reprotonated and Asp96 has deprotonated (in the N intermediate). An essential feature that made these experiments possible is the 75-fold signal enhancement through DNP. N(SB)-H correlations reveal that the newly deprotonated SB is accepting a hydrogen bond from an alcohol and C-C correlations show that Asp85 draws close to Thr89 before the primary proton transfer. Concurrently, N-C correlations between the SB and Asp85 show that helices C and G draw closer together just prior to the proton transfer and relax thereafter. Together, these results indicate that Thr89 serves to relay the SB proton to Asp85 and that creating this pathway involves rapprochement between the C and G helices as well as chromophore torsion.
Topics: Bacteriorhodopsins; Halobacterium salinarum; Ion Pumps; Light; Nuclear Magnetic Resonance, Biomolecular
PubMed: 29489362
DOI: 10.1021/jacs.8b00022 -
Pacific Symposium on Biocomputing.... 1998We have studied opto-electric properties of wild type bacteriorhodopsin and its two artificial variants. We have measured opto-electric responses with respect to...
We have studied opto-electric properties of wild type bacteriorhodopsin and its two artificial variants. We have measured opto-electric responses with respect to wavelength for all three proteins and we describe the use of the proteins for color detection. Opto-electric responses of proteins to set of colored lights were measured and it has been shown that bacteriorhodopsin and its variants can be used to recognize color. A simple equation for estimating opto-electric response to arbitrary spectrum is given.
Topics: Bacteriorhodopsins; Color; Darkness; Electrochemistry; Halobacterium salinarum; Light; Optics and Photonics; Pattern Recognition, Automated; Retinaldehyde; Schiff Bases; Spectrophotometry
PubMed: 9697209
DOI: No ID Found -
Journal of the American Chemical Society Nov 2014The folding mechanisms of helical membrane proteins remain largely uncharted. Here we characterize the kinetics of bacteriorhodopsin folding and employ φ-value analysis...
The folding mechanisms of helical membrane proteins remain largely uncharted. Here we characterize the kinetics of bacteriorhodopsin folding and employ φ-value analysis to explore the folding transition state. First, we developed and confirmed a kinetic model that allowed us to assess the rate of folding from SDS-denatured bacteriorhodopsin (bRU) and provides accurate thermodynamic information even under influence of retinal hydrolysis. Next, we obtained reliable φ-values for 16 mutants of bacteriorhodopsin with good coverage across the protein. Every φ-value was less than 0.4, indicating the transition state is not uniquely structured. We suggest that the transition state is a loosely organized ensemble of conformations.
Topics: Bacteriorhodopsins; Hydrolysis; Kinetics; Models, Molecular; Mutation; Protein Denaturation; Protein Folding; Sodium Dodecyl Sulfate; Thermodynamics
PubMed: 25369295
DOI: 10.1021/ja508359n -
Biochemistry. Biokhimiia Nov 2001The scheme of the bacteriorhodopsin photocycle associated with a transmembrane proton transfer and electrogenesis is considered. The role of conformational changes in... (Review)
Review
The scheme of the bacteriorhodopsin photocycle associated with a transmembrane proton transfer and electrogenesis is considered. The role of conformational changes in the polypeptide chain during the proton transport is discussed.
Topics: Bacteriorhodopsins; Electrochemistry; Photochemistry; Protons
PubMed: 11743867
DOI: 10.1023/a:1013127117712 -
The Journal of Chemical Physics Mar 2018Precisely quantifying the energetics that drive the folding of membrane proteins into a lipid bilayer remains challenging. More than 15 years ago, atomic force...
Precisely quantifying the energetics that drive the folding of membrane proteins into a lipid bilayer remains challenging. More than 15 years ago, atomic force microscopy (AFM) emerged as a powerful tool to mechanically extract individual membrane proteins from a lipid bilayer. Concurrently, fluctuation theorems, such as the Jarzynski equality, were applied to deduce equilibrium free energies (ΔG) from non-equilibrium single-molecule force spectroscopy records. The combination of these two advances in single-molecule studies deduced the free-energy of the model membrane protein bacteriorhodopsin in its native lipid bilayer. To elucidate this free-energy landscape at a higher resolution, we applied two recent developments. First, as an input to the reconstruction, we used force-extension curves acquired with a 100-fold higher time resolution and 10-fold higher force precision than traditional AFM studies of membrane proteins. Next, by using an inverse Weierstrass transform and the Jarzynski equality, we removed the free energy associated with the force probe and determined the molecular free-energy landscape of the molecule under study, bacteriorhodopsin. The resulting landscape yielded an average unfolding free energy per amino acid (aa) of 1.0 ± 0.1 kcal/mol, in agreement with past single-molecule studies. Moreover, on a smaller spatial scale, this high-resolution landscape also agreed with an equilibrium measurement of a particular three-aa transition in bacteriorhodopsin that yielded 2.7 kcal/mol/aa, an unexpectedly high value. Hence, while average unfolding ΔG per aa is a useful metric, the derived high-resolution landscape details significant local variation from the mean. More generally, we demonstrated that, as anticipated, the inverse Weierstrass transform is an efficient means to reconstruct free-energy landscapes from AFM data.
Topics: Bacteriorhodopsins; Lipid Bilayers; Microscopy, Atomic Force; Protein Folding; Thermodynamics
PubMed: 29604885
DOI: 10.1063/1.5009108 -
Molecular Microbiology Jun 1998The archaeal rhodopsins are a family of seven-transmembrane-helix, visual pigment-like proteins found in Halobacterium salinarum and related halophilic Archaea. Two,... (Review)
Review
The archaeal rhodopsins are a family of seven-transmembrane-helix, visual pigment-like proteins found in Halobacterium salinarum and related halophilic Archaea. Two, bacteriorhodopsin (BR) and halorhodopsin (HR), are transport rhodopsins that carry out light-driven electrogenic translocation of protons and chloride, respectively, across the cell membrane. The other two, sensory rhodopsins I and II (SRI and SRII), are phototaxis receptors that send signals to tightly bound transducer proteins that in turn control a phosphorylation cascade modulating the cell's flagellar motors. Recent progress has cast light on how nature has modified the common design of these proteins to carry out their distinctly different functions: electrogenic ion transport and non-electrogenic signal transduction. A key shared mechanism between BR and SRII appears to be an interhelical salt bridge locked conformational switch that is released by photoisomerization of retinal. In BR disruption of the lock opens a cytoplasmic half-channel that ensures uptake of the transported proton from the cytoplasmic side of the membrane at a critical time in the pumping cycle. Transducer-free SRI uses the same mechanism to carry out light-driven proton transport, but interaction with its transducer blocks the cytoplasmic half-channel thereby interrupting the transport cycle. In SRI, transducer interaction also disrupts the salt bridge in the dark, poising the receptor in an intermediate conformation able to produce opposite signals depending on the colour of the stimulus light. A model for signalling is proposed in which the salt bridge-controlled half-channel is used to modulate interaction with the Htr proteins when the receptor signalling states are formed.
Topics: Archaea; Archaeal Proteins; Bacteriorhodopsins; Carotenoids; Halorhodopsins; Ion Transport; Sensory Rhodopsins; Signal Transduction
PubMed: 9680197
DOI: 10.1046/j.1365-2958.1998.00859.x -
Biochemistry. Biokhimiia Nov 2001This review begins with a brief history of early studies on the involvement of lipids in certain bacteriorhodopsin (BR) properties. Such properties include the... (Review)
Review
This review begins with a brief history of early studies on the involvement of lipids in certain bacteriorhodopsin (BR) properties. Such properties include the regulation of the pK for the purple to blue transition caused by deionization, and the reformation of trimers from monomers after exposure of the purple membrane to Triton X-100. Most of the review is devoted to newer studies which indicate an important role for the neutral lipid squalene in the functional stability of the fast-decaying M-intermediate, for its decay through a pathway involving the O-intermediate, and for the regulation of the relative amounts of slow-decaying and fast-decaying forms of M. Participation of a peripheral acidic amino acid in the overall expression of fast-decaying M is also discussed. Initial studies suggest that the acidic amino acid may be Asp36 and/or Asp38.
Topics: Bacteriorhodopsins; Lipids; Structure-Activity Relationship
PubMed: 11743875
DOI: 10.1023/a:1013143621346 -
Biophysical Journal Jul 2003We have investigated the role of the native lipids on bacteriorhodopsin (bR) proton transfer and their connection with the cation-binding role. We observe that both the...
We have investigated the role of the native lipids on bacteriorhodopsin (bR) proton transfer and their connection with the cation-binding role. We observe that both the efficiency of M formation and the kinetics of M rise and decay depend on the lipids and lattice but, as the lipids are removed, the cation binding is a much less important factor for the proton pumping function. Upon 75% delipidation using 3-[(cholamidopropyl)dimethylammonio]-propanesulfonate (CHAPS), the M formation and decay kinetics are much slower than the native, and the efficiency of M formation is approximately 30%-40% that of the native. Upon monomerization of bR by Trition X-100, the efficiency of M recovers close to that of the native (depending on pH), M formation is approximately 10 times faster, and M decay kinetics are comparable to native at pH 7. The same results on the M intermediate are observed if deionized blue bR (deI bbR) is treated with these detergents (with or without pH buffers present), even though deionized blue bR containing all the lipids has no photocycle. This suggests that the cation(s) has a role in native bR that is different than in delipidated or monomerized bR, even so far as to suggest that the cation(s) becomes unimportant to the function as the lipids are removed.
Topics: Absorptiometry, Photon; Bacteriorhodopsins; Biological Transport; Ions; Kinetics; Lipids; Photochemistry; Protein Conformation; Proton Pumps; Protons; Spectrum Analysis
PubMed: 12829497
DOI: 10.1016/S0006-3495(03)74487-7 -
Biochemistry. Biokhimiia Nov 2001In the last few years, detailed structural information from high-resolution x-ray diffraction has been added to the already large body of spectroscopic and mutational... (Review)
Review
In the last few years, detailed structural information from high-resolution x-ray diffraction has been added to the already large body of spectroscopic and mutational data on the bacteriorhodopsin proton transport cycle. Although there are still many gaps, it is now possible to reconstruct the main events in the translocation of the proton and how they are coupled to the photoisomerization of the retinal chromophore. Future structural work will concentrate on describing the details of the individual proton transfer steps during the photocycle.
Topics: Bacteriorhodopsins; Crystallography, X-Ray; Models, Molecular; Protein Conformation
PubMed: 11743864
DOI: 10.1023/a:1013159532733