The Ca2+ binding to deionized monomerized and to retinal removed bacteriorhodopsin

Yang, Difei; El‐Sayed, Mostafa A.

doi:10.1016/s0006-3495(95)80075-5

Cited by 21 publications

(24 citation statements)

References 30 publications

(27 reference statements)

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“…The observation of only one type of binding site for bO is consistent with recent results (41) in which it was observed that the binding characteristics of the two high-affinity sites of bR are altered upon retinal cleavage. Specifically, only one highaffinity site is observed in bO, which exhibits an apparent binding constant intermediate between the first and second high-affinity binding sites in deionized bR.…”

Section: Discussionsupporting

confidence: 91%

Detection of a Yb ³⁺ binding site in regenerated bacteriorhodopsin that is coordinated with the protein and phospholipid head groups

Roselli¹,

Boussac²,

Mattioli³

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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Near infrared Yb 3؉ vibronic sideband spectroscopy was used to characterize specific lanthanide binding sites in bacteriorhodopsin (bR) and retinal free bacteriorhodopsin (bO). The VSB spectra for deionized bO regenerated with a ratio of 1:1 and 2:1 ion to bO are identical. Application of a two-dimensional anti-correlation technique suggests that only a single Yb 3؉ site is observed. The Yb 3؉ binding site in bO is observed to consist of PO 2 ؊ groups and carboxylic acid groups, both of which are bound in a bidentate manner. An additional contribution most likely arising from a phenolic group is also observed. This implies that the ligands for the observed single binding site are the lipid head groups and amino acid residues. The vibronic sidebands of Yb 3؉ in deionized bR regenerated at a ratio of 2:1 ion to bR are essentially identical to those in bO. The other high-affinity binding site is thus either not evident or its f luorescence is quenched. A discussion is given on the difference in binding of Ca 2؉ (or Mg 2؉ ) and lanthanides in phospholipid membrane proteins.Bacteriorhodopsin (bR) is the only protein in the purple membrane of the halophilic archaebacterium Halobacterium salinarium. This protein is capable of light-driven transmembrane-proton translocation during a specific photochemical cycle (1-3) involving a retinal chromophore in a protonated Schiff base linkage with the lysine residue Lys-216 (4-6).The binding of metal cations, specifically Ca 2ϩ and Mg 2ϩ , is now recognized as essential for the proper functioning of the proton pumping mechanism of bR (7,8). Removal of the cations by deionization or acidification shifts the absorption maximum of purple membrane to 604 nm (blue bR) (9, 10). The absorption maximum of native bR (560-570 nm) returns upon the addition of different metal cations (9, 10). The initial step in the bR photocycle involves the excitation of the retinal by absorption of a photon (11-15). This step is followed by isomerization of the initially all-trans retinal to form the 13-cis retinal (11-15). Thermal relaxation leads to several intermediates, the net result being translocation of a proton from the cytoplasmic to the extracellular side of the membrane. Eventually, bR returns to its ground state from which it can undergo another photocycle (11-15). While retinal photoisomerization takes place in blue bR, the M 412 intermediate is not formed and, thus, proton pumping does not occur (9, 10).The locations of the cation binding sites are still not well known. Stroud and colleagues (16,17) have observed several Pb 2ϩ binding sites in bR via x-ray diffraction. The ligands that bind the cations cannot be identified from such studies due to the present inability to form three-dimensional crystals of bR. To understand the role that these metal cations play in the function of bR, it is necessary to know the location of the cations in the protein and the chemical nature of the ligands that bind them. For Ca 2ϩ binding proteins, such as bR, the latter is difficult to probe becau...

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Section: Discussionsupporting

confidence: 91%

Detection of a Yb ³⁺ binding site in regenerated bacteriorhodopsin that is coordinated with the protein and phospholipid head groups

Roselli¹,

Boussac²,

Mattioli³

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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“…Two high-affinity sites were found for Ca 2ϩ to bind to deionized bR as well as four to six lower-affinity sites (Zhang et al, 1992), and removal of retinal removes one of the strongly bound cations (Yang and El-Sayed, 1995). A number of groups have suggested that there is at least one specific internal binding site close to retinal and has binding constants dependent on the cation (Jonas and Ebrey, 1991;Ariki and Lanyi, 1986;Zhang et al, 1992).…”

Section: Introductionmentioning

confidence: 99%

Fourier Transform Infrared Study of the Effect of Different Cations on Bacteriorhodopsin Protein Thermal Stability

Heyes

Wang²,

Sanii

et al. 2002

Biophysical Journal

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The effect of divalent ion binding to deionized bacteriorhodopsin (dI-bR) on the thermal transitions of the protein secondary structure have been studied by using temperature-dependent Fourier transform infrared (FT-IR) spectroscopy. The native metal ions in bR, Ca(2+), and Mg(2+), which we studied previously, are compared with Mn(2+), Hg(2+), and a large, synthesized divalent organic cation, ((Et)(3)N)(2)Bu(2+). It was found that in all cases of ion regeneration, there is a pre-melting, reversible conformational transition in which the amide frequency shifts from 1665 to 1652 cm(-1). This always occurs at approximately 80 degrees C, independent of which cation is used for the regeneration. The irreversible thermal transition (melting), monitored by the appearance of the band at 1623 cm(-1), is found to occur at a lower temperature than that for the native bR but higher than that for acid blue bR in all cases. However, the temperature for this transition is dependent on the identity of the cation. Furthermore, it is shown that the mechanism of melting of the organic cation regenerated bR is different than for the metal cations, suggesting a difference in the type of binding to the protein (either to different sites or different binding to the same site). These results are used to propose specific direct binding mechanisms of the ions to the protein of deionized bR.

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“…One of these binding sites is associated with the blue to purple color transition (18). Only one strongly bound Ca 2ϩ ion was found after removing the retinal chromophore (23), indicating that one of the high affinity sites might not be far from the retinal. The presence of a divalent cation near Asp85 has been suggested to keep it in the ionized form and could be responsible for Figure 1.…”

Section: Introductionmentioning

confidence: 99%

The Effect of Metal Cation Binding on the Protein, Lipid and Retinal Isomeric Ratio in Regenerated Bacteriorhodopsin of Purple Membrane¶

Wang

El‐Sayed

2001

Photochem Photobiol

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The effect of metal cation binding on bacteriorhodopsin (bR) in purple membrane has been examined using in situ attenuated total reflection-Fourier transform infrared difference spectroscopy in aqueous media. It is known that adding metal cations to deionized bR regenerates the purple state from its blue state and recovers the proton pump function. During this process, infrared spectral changes in the frequency region of 1800-1000 cm-1 are monitored. The results reveal that metal cation binding affects the protein conformation, the retinal isomeric composition as well as lipid head groups. It is also observed that metal cation binding induces conformational changes in the alpha 1-helix region of bR, converting the portion of its alpha 1-helical domain into beta-turn or disordered coil. In addition, the influence of Ho3+ binding on the protein and lipid is observed to be larger than that of Ca2+. These results suggest that some of the metal cation binding sites are on the membrane lipid domain, while others could be on the intrahelical domain or interhelical loops where the Asp and Glu are located (binding with their COO- groups). Our results also suggest that the removal of the C-terminal of bR increase the accessibility of the binding site of metal cations, which affects protein conformational structure. All these observations are discussed in terms of the two proposals given in the literature regarding the metal cation binding sites.

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The Ca2+ binding to deionized monomerized and to retinal removed bacteriorhodopsin

Cited by 21 publications

References 30 publications

Detection of a Yb ³⁺ binding site in regenerated bacteriorhodopsin that is coordinated with the protein and phospholipid head groups

Detection of a Yb ³⁺ binding site in regenerated bacteriorhodopsin that is coordinated with the protein and phospholipid head groups

Fourier Transform Infrared Study of the Effect of Different Cations on Bacteriorhodopsin Protein Thermal Stability

The Effect of Metal Cation Binding on the Protein, Lipid and Retinal Isomeric Ratio in Regenerated Bacteriorhodopsin of Purple Membrane¶

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The Ca2+ binding to deionized monomerized and to retinal removed bacteriorhodopsin

Cited by 21 publications

References 30 publications

Detection of a Yb 3+ binding site in regenerated bacteriorhodopsin that is coordinated with the protein and phospholipid head groups

Detection of a Yb 3+ binding site in regenerated bacteriorhodopsin that is coordinated with the protein and phospholipid head groups

Fourier Transform Infrared Study of the Effect of Different Cations on Bacteriorhodopsin Protein Thermal Stability

The Effect of Metal Cation Binding on the Protein, Lipid and Retinal Isomeric Ratio in Regenerated Bacteriorhodopsin of Purple Membrane¶

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Detection of a Yb ³⁺ binding site in regenerated bacteriorhodopsin that is coordinated with the protein and phospholipid head groups

Detection of a Yb ³⁺ binding site in regenerated bacteriorhodopsin that is coordinated with the protein and phospholipid head groups