The binding site of the strongly bound Eu<sup>3+</sup> in Eu<sup>3+</sup>‐regenerated bacteriorhodopsin

Sweetman, L.; El‐Sayed, Mostafa A.

doi:10.1016/0014-5793(91)80531-7

Cited by 18 publications

(17 citation statements)

References 42 publications

(12 reference statements)

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“…2B shows that the Eu 3+ standards in the chelating solution obey Beer's law in the range 0–2 μM, which is the range used for the experiment. These results are in agreement with previously published work on Eu 3+ assays [24,25].…”

Section: Resultssupporting

confidence: 94%

“…We then decided to use the strong emitting nature of Eu 3+ chelates to investigate the effects of cation binding to bR upon delipidation. This sensitive fluorescence method has been used previously [24,25] to assay specific detection of free Eu 3+ in equilibrium with membrane‐bound Eu 3+ in order to calculate their binding constants to the deionized blue membrane. Aqueous Eu 3+ in the presence of the chelating agent TTA in TOPO has an excitation and emission spectrum as shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The concentration of Eu 3+ bound to the bR is found using the methods adapted previously [24,25]. Briefly, a chelating agent used to detect the free Eu 3+ fluorescence in the supernatants of the Eu 3+ regenerated bR solutions.…”

Section: Methodsmentioning

confidence: 99%

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Eu³⁺ binding to europium‐regenerated bacteriorhodopsin upon delipidation and monomerization

Heyes¹,

Reynolds²,

El‐Sayed³

2004

FEBS Letters

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We have studied the e¡ect of monomerization of the purple membrane lattice, as well as removal of 75% of the lipids, on the binding properties of Eu 3+ ions. We found that delipidation and monomerization do not cause the cations to lose their binding ability to the protein. This suggests that the three most strongly bound Eu 3+ cations do not bind to the lipids, but directly bind to the protein. Furthermore, we found that delipidation actually causes a slight increase in the binding a⁄nity. This is likely a result of reduced aggregation of europium-regenerated bacteriorhodopsin (bR) upon lipid removal causing more exposure of the binding sites to the Eu 3+ cations. These results, taken with those from our previous publication [Heyes and El-Sayed, Biophys. J. 85 (2003)

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Section: Resultssupporting

confidence: 94%

Section: Resultsmentioning

confidence: 99%

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Eu³⁺ binding to europium‐regenerated bacteriorhodopsin upon delipidation and monomerization

Heyes¹,

Reynolds²,

El‐Sayed³

2004

FEBS Letters

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“…Mutation studies of Ca 2ϩ binding observed a large change in the binding constant upon replacement of D85 and D212 by neutral amino acid residues (22,23). The studies on Eu 3ϩ emission and its binding results suggest that Eu 3ϩ is not far from the retinal to allow efficient quenching of its emission (25). More recently, Fourier transform infrared (FTIR) studies (26) showed that a vibrational band resulting from the interaction of the aspartate group (COO Ϫ ) and Ca 2ϩ , while present in bR, disappears in the D85N mutant, as well as in deionized bR.…”

mentioning

confidence: 99%

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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“…Both the PSB and at least one (Asp-85) of the two aspartates (or all according to the mIR studies [36]) cannot bc the candidates, as they lose their charge during the appcarance of the transient absorption at 296 nm. Recently, a metal cation is proposed to be also present within the retinal pocket [48,49]. The removal of tnetal cations is found to eliminate both the M412 formation as well as the 296 nm absorption [45].…”

Section: IImentioning

confidence: 99%

The use of tryptophan mutants in identifying the 296 nm transient absorbing species during the photocycle of bacteriorhodopsin

Jang

El‐Sayed

et al. 1991

FEBS Letters

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The transient absorption at 296 nm was part of the spectroscopic evidence that initiated the proposal that tyrosinate (Tyr-) is formed during, and important to, the photocycle of bacteriorhodopsin (bR). Recent evidence against such a proposal comes from the results of NMR, UV Raman as well as electron cryo-microscopic structural studies. This makes it credible to assign this absorption to a charge perturbation of the lowest energy absorption of one of the tryptophan (Trp) residues in bR. The transient absorption at 296 nm is examined for each of 8 tryptophan mutants in which Trp is substituted by phenylalanine or cysteine, which absorb at shorter wavelength. It is shown that while all go through the photocycle, all but Trp-182 mutant show this transient absorption. This strongly suggests the assignment of this absorption to a charge perturbaton of the lowest energy absorption of Trp-I 82 during the photocycle. The chemical identity of the perturbing charge(s) is briefly discussed.

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The binding site of the strongly bound Eu³⁺ in Eu³⁺‐regenerated bacteriorhodopsin

Cited by 18 publications

References 42 publications

Eu³⁺ binding to europium‐regenerated bacteriorhodopsin upon delipidation and monomerization

Eu³⁺ binding to europium‐regenerated bacteriorhodopsin upon delipidation and monomerization

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

The use of tryptophan mutants in identifying the 296 nm transient absorbing species during the photocycle of bacteriorhodopsin

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The binding site of the strongly bound Eu3+ in Eu3+‐regenerated bacteriorhodopsin

Cited by 18 publications

References 42 publications

Eu3+ binding to europium‐regenerated bacteriorhodopsin upon delipidation and monomerization

Eu3+ binding to europium‐regenerated bacteriorhodopsin upon delipidation and monomerization

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

The use of tryptophan mutants in identifying the 296 nm transient absorbing species during the photocycle of bacteriorhodopsin

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The binding site of the strongly bound Eu³⁺ in Eu³⁺‐regenerated bacteriorhodopsin

Eu³⁺ binding to europium‐regenerated bacteriorhodopsin upon delipidation and monomerization

Eu³⁺ binding to europium‐regenerated bacteriorhodopsin upon delipidation and monomerization

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