Sensitive Detection of Protein−Lipid Interaction Change on Bacteriorhodopsin Using Dodecyl β-<scp>d</scp>-Maltoside

Sasaki, Takanori; Demura, Makoto; Kato, Norihiro; Mukai, Yukinori

doi:10.1021/bi101993s

Cited by 12 publications

(12 citation statements)

References 56 publications

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“…However, the crystal structure of NpHR produced in E. coli has not been revealed. BR also forms a homotrimer in the membrane (2,6,7), however the trimeric structure dissociates into monomers when detergent has solubilized it (25)(26)(27).…”

Section: Introductionmentioning

confidence: 99%

Homotrimer Formation and Dissociation of pharaonis Halorhodopsin in Detergent System

Tsukamoto

Sasaki

Fujimoto

et al. 2012

Biophysical Journal

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Halorhodopsin from NpHR is a light-driven Cl(-) pump that forms a trimeric NpHR-bacterioruberin complex in the native membrane. In the case of NpHR expressed in Escherichia coli cell, NpHR forms a robust homotrimer in a detergent DDM solution. To identify the important residue for the homotrimer formation, we carried out mutation experiments on the aromatic amino acids expected to be located at the molecular interface. The results revealed that Phe(150) was essential to form and stabilize the NpHR trimer in the DDM solution. Further analyses for examining the structural significance of Phe(150) showed the dissociation of the trimer in F150A (dimer) and F150W (monomer) mutants. Only the F150Y mutant exhibited dissociation into monomers in an ionic strength-dependent manner. These results indicated that spatial positions and interactions between F150-aromatic side chains were crucial to homotrimer stabilization. This finding was supported by QM calculations. In a functional respect, differences in the reaction property in the ground and photoexcited states were revealed. The analysis of photointermediates revealed a decrease in the accumulation of O, which is important for Cl(-) release, and the acceleration of the decay rate in L1 and L2, which are involved in Cl(-) transfer inside the molecule, in the trimer-dissociated mutants. Interestingly, the affinity of them to Cl(-) in the photoexcited state increased rather than the trimer, whereas that in the ground state was almost the same without relation to the oligomeric state. It was also observed that the efficient recovery of the photocycle to the ground state was inhibited in the mutants. In addition, a branched pathway that was not included in Cl(-) transportation was predicted. These results suggest that the trimer assembly may contribute to the regulation of the dynamics in the excited state of NpHR.

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Section: Introductionmentioning

confidence: 99%

Homotrimer Formation and Dissociation of pharaonis Halorhodopsin in Detergent System

Tsukamoto

Sasaki

Fujimoto

et al. 2012

Biophysical Journal

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“…The systems under study are lipidic phases from the industrially produced dodecyl β -maltoside ( β Mal-C 12 ), a glycolipid which has been used in the purification and stabilization of proteins, like RNA polymerase, and the detection of protein-lipid interactions3839. The maltoside sugar is sufficiently large for the fluorescence molecule to be probed in the head group region22.…”

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confidence: 99%

Detecting local heterogeneity and ionization ability in the head group region of different lipidic phases using modified fluorescent probes

Abou‐Zied

Zahid

Khyasudeen

et al. 2015

Sci Rep

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Local heterogeneity in lipid self-assembly is important for executing the cellular membrane functions. In this work, we chemically modified 2-(2′-hydroxyphenyl)benzoxazole (HBO) and attached a C8 alkyl chain in two different locations to probe the microscopic environment of four lipidic phases of dodecyl β-maltoside. The fluorescence change in HBO and the new probes (HBO-1 and HBO-2) shows that in all phases (micellar, hexagonal, cubic and lamellar) three HBO tautomeric species (solvated syn-enol, anionic, and closed syn-keto) are stable. The formation of multi tautomers reflects the heterogeneity of the lipidic phases. The results indicate that HBO and HBO-1 reside in a similar location within the head group region, whereas HBO-2 is slightly pushed away from the sugar-dominated area. The stability of the solvated syn-enol tautomer is due to the formation of a hydrogen bond between the OH group of the HBO moiety and an adjacent oxygen atom of a sugar unit. The detected HBO anions was proposed to be a consequence of this solvation effect where a hydrogen ion abstraction by the sugar units is enhanced. Our results point to a degree of local heterogeneity and ionization ability in the head group region as a consequence of the sugar amphoterism.

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“…Different phases of a synthetic lipid were used as model systems to study the local heterogeneity in the head group region . The lipidic material was from the industrially produced dodecyl β ‐maltoside ( β Mal‐C 12 ), a glycolipid that has been used in the purification and stabilization of proteins, like RNA polymerase, and the detection of protein–lipid interactions . The micellar, hexagonal, cubic ( Ia3d space group) and lamellar phases are all in normal (type I) state and were obtained at a specific concentration and temperature of aqueous formulation of β Mal‐C 12 , as reported in its phase diagram .…”

Section: Probing Lipid Self‐assemblymentioning

confidence: 99%

Photophysical properties of hydroxyphenyl benzazoles and their applications as fluorescent probes to study local environment in DNA, protein and lipid

2016

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Fluorescence techniques have drawn increasing attention because they provide crucial information about molecular interactions in protein-ligand systems beyond that obtained by other methods. The advantage of fluorescence spectroscopy stems from the fact that the majority of molecules in biological systems do not exhibit fluorescence, making fluorescent probes useful with high sensitivity. Also, the fluorescence emission is highly sensitive to the local environment, providing a valuable tool to investigate the nature of binding sites in macromolecules. In this review, we discuss some of the important applications of a class of molecules that have been used as fluorescent probes in a variety of studies. Hydroxyphenyl benzazoles (HBXs) show distinct spectroscopic features that make them suitable probes for the study of certain biological mechanisms in DNA, protein and lipid. In particular, the complex photophysics of 2-(2'-hydroxyphenyl)benzoxazole (HBO) and the distinguished fluorescence signatures of its different tautomeric forms make this molecule a useful probe in several applications. Among these are probing the DNA local environment, study of the flexibility and specificity of protein-binding sites, and detecting the heterogeneity and ionization ability of the head groups of different lipidic phases. The spectroscopy of HBX molecules and some of their chemically modified structures is also reviewed.

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Sensitive Detection of Protein−Lipid Interaction Change on Bacteriorhodopsin Using Dodecyl β-d-Maltoside

Cited by 12 publications

References 56 publications

Homotrimer Formation and Dissociation of pharaonis Halorhodopsin in Detergent System

Homotrimer Formation and Dissociation of pharaonis Halorhodopsin in Detergent System

Detecting local heterogeneity and ionization ability in the head group region of different lipidic phases using modified fluorescent probes

Photophysical properties of hydroxyphenyl benzazoles and their applications as fluorescent probes to study local environment in DNA, protein and lipid

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