Stability Of Bacteriorhodopsin Research Articles

Influence of osmolytes and ionic liquids on the Bacteriorhodopsin structure in the absence and presence of oxidative stress: A combined experimental and computational study

Understanding the folding and stability of membrane proteins is of great importance in protein science. Recently, osmolytes and ionic liquids (ILs) are increasingly being used as drug delivery systems in the biopharmaceutical industry. However, the stability of membrane proteins in the presence of osmolytes and ILs is not yet fully understood. Besides, the effect of oxidative stress on membrane proteins with osmolytes or ILs has not been investigated. Therefore, we studied the influence of osmolytes and ILs as co-solvents on the stability of a model membrane protein (i.e., Bacteriorhodopsin in purple membrane of Halobacterium salinarum), using UV–Vis spectroscopy and molecular dynamics (MD) simulations. The MD simulations allowed us to determine the flexibility and solvent accessible surface area (SASA) of Bacteriorhodopsin protein in the presence and/or absence of co-solvents, as well as to carry out principal component analysis (PCA) to identify the most important movements in this protein. In addition, by means of UV–Vis spectroscopy we studied the effect of oxidative stress generated by cold atmospheric plasma on the stability of Bacteriorhodopsin in the presence and/or absence of co-solvents. This study is important for a better understanding of the stability of proteins in the presence of oxidative stress.

1B1612 人工リン脂質2層膜に再構成されたバクテリオロドプシンの分子集合・構造安定性における古細菌型脂質の役割(膜蛋白質1,第49回日本生物物理学会年会)

1B1612 人工リン脂質2層膜に再構成されたバクテリオロドプシンの分子集合・構造安定性における古細菌型脂質の役割(膜蛋白質1,第49回日本生物物理学会年会)

3P119 アシル鎖長の異なる3種類の脂質膜リポソームに再構成されたバクテリオロドプシンの分子集合状態の変化が引き起こす分子構造・構造安定性への影響(膜蛋臼質,第48回日本生物物理学会年会)

3P119 アシル鎖長の異なる3種類の脂質膜リポソームに再構成されたバクテリオロドプシンの分子集合状態の変化が引き起こす分子構造・構造安定性への影響(膜蛋臼質,第48回日本生物物理学会年会)

Comparison of Stability Predictions and Simulated Unfolding of Rhodopsin Structures†

Developing a better mechanistic understanding of membrane protein folding is urgently needed because of the discovery of an increasing number of human diseases, where membrane protein instability and misfolding is involved. Towards this goal, we investigated folding and stability of 7-transmembrane (TM) helical bundles by computational methods. We compared the results of three different algorithms for predicting changes in stability of proteins against an experimental mutation dataset obtained for bacteriorhodopsin (BR) and mammalian rhodopsin and find that 61.6% and 70.6% of the mutation results can potentially be explained by known local contributors to the stability of the folded state of BR and mammalian rhodopsin, respectively. To obtain further information on the predicted folding pathway of 7-TM proteins, we conducted simulated thermal unfolding experiments of all available rhodopsin structures with resolution better than 3 angstroms using the Floppy Inclusions and Rigid Substructure Topography (FIRST) method (Jacobs, D. J., A. J. Rader, L. A. Kuhn and M. F. Thorpe [2001] Proteins 44, 150) described previously for a single mammalian rhodopsin structure (Rader et al. [2004] PNAS 101, 7246). In statistical comparison we found that structures of mammalian rhodopsin have a stability core that is characterized by long-range interactions involving amino acids close in space but distant in sequence comprising positions from both extracellular loop and TM regions. In contrast, BR-simulated unfolding does not reveal such a core but is dominated by interactions within individual and groups of TM helices, consistent with the two-stage hypothesis of membrane protein folding. Similar results were obtained for halo- and sensory rhodopsins as for BRs. However, the average folding core energies of sensory rhodopsins were in between those observed for mammalian rhodopsins and BRs hinting at a possible evolution of these structures toward a rhodopsin-like behavior. These results support the conclusion that although the two-stage model can explain the mechanisms of folding and stability of BR, it fails to account for the folding and stability of mammalian rhodopsin, even though the two proteins are structurally related.

バクテリオロドプシン光誘起変性に見られた構造安定性の不均一性に対する溶媒pHの効果

バクテリオロドプシン光誘起変性に見られた構造安定性の不均一性に対する溶媒pHの効果

Effect of Asp85 replacement by Thr on the conformation, surface electric properties and stability of bacteriorhodopsin

Abstract The role of Asp85 in maintaining bacteriorhodopsin structure has been analyzed by infrared spectroscopy, electric light scattering and differential scanning calorimetry. In comparison with the wild type bacteriorhodopsin, the mutant protein D85T shows a different conformation, electric dipole moments and decreased thermal stability. The conformational rearrangements affect both the transmembrane helices and the extramembranous protein segments. Both electric dipoles—the permanent dipole moment and the electric polarizability—have drastically lower values for the membranes containing D85T variant of bacteriorhodopsin. Therefore, this single amino acid mutation not only changes bacteriorhodopsin function, converting the protein into a halorhodopsin-like chloride pump, but it also alters its overall conformation and surface electric state.

Structure and function in bacteriorhodopsin: the role of the interhelical loops in the folding and stability of bacteriorhodopsin

Bacteriorhodopsin functions as a light-driven proton pump in Halobacterium salinarium. The functional protein consists of an apoprotein, bacterioopsin, with seven transmembrane α helices together with a covalently bound all-trans retinal chromophore. In order to study the role of the interhelical loop conformations in the structure and function of bacteriorhodopsin, we have constructed bacterioopsin genes where each loop is replaced, one at a time, by a peptide linker consisting of Gly-Gly-Ser-repeat sequences, which are believed to have flexible conformations. These mutant proteins have been expressed in Escherichia coli, purified and reconstituted with all-trans retinal in l-α-1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/3-(3-cholamidopropyl)dimethylammonio-1-propane sulfonate (CHAPS)/SDS and l-α-1,2-dihexanoylphosphatidylcholine (DHPC)/DMPC/SDS micelles. Wild-type-like chromophore formation was observed in all the mutants containing single loop replacements. In the BC and FG mutants, an additional chromophore band with an absorption band at about 480 nm was observed, which was in equilibrium with the 550 nm, wild-type band. The position of the equilibrium depended on temperature, SDS and relative DMPC concentration. The proton pumping activity of all of the mutants was comparable to that of wild-type bR except for the BC and FG mutants, which had lower activity. All of the loop mutants were more sensitive to denaturation by SDS than the wild-type protein, except the mutant where the DE loop was replaced. These results suggest that a specific conformation of all the loops of bR, except the DE loop, contributes to bR stability and is required for the correct folding and function of the protein. An increase in the relative proportion of DHPC in DHPC/DMPC micelles, which reduces the micelle rigidity and alters the micelle shape, resulted in lower folding yields of all loop mutants except the BC and DE mutants. This effect of micelle rigidity on the bR folding yield correlated with a loss in stability of a partially folded, seven-transmembrane apoprotein intermediate state in SDS/DMPC/CHAPS micelles. The folding yield and stability of the apoprotein intermediate state both decreased for the loop mutants in the order WT∼BC∼DE>FG>AB>EF⩾CD, where the EF and CD loop mutants were the least stable.

3P085n-nonyl-β-glucosideにより可溶化されたバクテリオロドプシンの構造安定性

3P085n-nonyl-β-glucosideにより可溶化されたバクテリオロドプシンの構造安定性

Bleaching of bacteriorhodopsin by continuous light

A new two step photobleaching process is observed under continuous illumination of bacteriorhodopsin. This photobleaching is considerable even at physiological temperatures and becomes large at 50–60°C. The photobleaching also increases with increasing pH from 7 to 10. We suggest that the bleaching at its final stage could be due to the dissociation of the retinal and a local thermal denaturation-like process. These facts may question the generally held belief that BR is a stable protein in vivo for a long period of time. Our results may have relevance also to practical applications of bacteriorhodopsin where the stability of bacteriorhodopsin is a key issue. In certain instances, the use of bacteriorhodopsin may require cooled conditions. Here, we defined the conditions under which bacteriorhodopsin is stable. The permanent photobleaching offers a new way of picture imaging and information input for bacteriorhodopsin-based optical devices.

Correlation between surfactant/micelle structure and the stability of bacteriorhodopsin in solution

The rate of solubilization and isothermal bleaching of bacteriorhodopsin (bR) in a series of nine alkylammonium surfactants is studied by using time-resolved optical spectroscopy. The surfactant series RN(+)R'(3) covers a range in tail length (R = C(12)H(25), C(14)H(29), or C(16)H(33)) and headgroup size and hydrophobicity (R' = CH(3); C(2)H(5), or C(3)H(7)). The rate of bleaching increases initially with increasing surfactant concentration but decreases at higher concentrations. Possible explanations for this behavior are discussed. The kinetic data are consistent with the penetration of the surfactant into the protein interior. Interaction of the surfactants with the protein is a complicated, multistep process, and the rate curves are a function of at least four variables: 1) the micellar environment, 2) the length of the surfactant tail, 3) the size of the headgroup, and 4) the hydrophobicity of the headgroup. Our data provide new insights into the molecular characteristics that help define the performance of surfactants in the solubilization and denaturation of membrane-bound proteins.