Nature of the individual Ca2+ binding sites in Ca2+-regenerated bacteriorhodopsin

Zhang, Y.N.; Sweetman, L.; Awad, Elias S.; El‐Sayed, Mostafa A.

doi:10.1016/s0006-3495(92)81929-x

Cited by 55 publications

(93 citation statements)

References 27 publications

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“…The binding experiment was carried out at room temperature as described in our previous paper (31 Thus the direction of the shift (i.e., red or blue) upon deionization is sensitive to whether the residue replaced is charged or neutral. Furthermore, replacement of the neutral residue preserved the direction of the absorption shift as in bR while the replacement of charged residues did not.…”

Section: Methodsmentioning

confidence: 99%

“…The essential point of this model is that cations affect the color transition only by changing the surface pH. The fact that deionization no longer has an effect on the spectrum of the purple membrane once the acidic lipids are removed or exchanged for neutral lipids gave strong evidence for this model (18) and its implication that metal cations are bound to the negatively charged surface of the purple membrane patch bR via nonspecific electrostatic attraction.Another model proposed by several other groups (21-32) emphasizes the existence of specific binding between some of the metal cations and negatively charged groups (e.g., carboxylate groups of Asp and Glu side chains) inside the protein, without excluding possible binding of some cations on the membrane surface (12,18,31). It was proposed (6,(22)(23)(24)32) that protonation changes near the Schiff base are directly linked to the binding of cations to the bR molecule.…”

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confidence: 99%

“…Another model proposed by several other groups (21-32) emphasizes the existence of specific binding between some of the metal cations and negatively charged groups (e.g., carboxylate groups of Asp and Glu side chains) inside the protein, without excluding possible binding of some cations on the membrane surface (12,18,31). It was proposed (6,(22)(23)(24)32) that protonation changes near the Schiff base are directly linked to the binding of cations to the bR molecule.…”

mentioning

confidence: 99%

“…It was proposed (6,(22)(23)(24)32) that protonation changes near the Schiff base are directly linked to the binding of cations to the bR molecule. Evidence (31)(32)(33) has supported the direct involvement of at least one cation in a special binding site, which would aid in maintaining the ionization state of key amino acids in the active site and thereby control the color of the purple membrane.…”

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confidence: 99%

“…To characterize the cation binding sites, various methods [e.g., electron spin resonance spectroscopy for Mn2+ (26,28), filtration techniques for Ca2+ and Hg2+ (27) (31) two highaffinity and four to six low-affinity Ca2+ binding sites in deionized blue membrane suspended in deionized, distilled water. We determined the association constants for the two most tightly bound Ca2W to be K1 = 0.63 j.M-1 and K2 = 0.14 ,uM-1, respectively at pH 4.…”

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Effects of genetic replacements of charged and H-bonding residues in the retinal pocket on Ca2+ binding to deionized bacteriorhodopsin.

Zhang

El‐Sayed

Bonet

et al. 1993

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

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Metal cations are known to be required for proton pumping by bacteriorhodopsin (bR). Previous studies found that bR has two high-affinity and four to six low-affinity Ca2+-binding sites. In our efforts to find the location of these Ca2+ sites, the effects of replacing charged (Asp-85, Asp-212, and Arg-82) and H-bonding (Tyr-185) residues in the retinal pocket on the color control and binding affinity of Ca2+ ions in Ca2+-regenerated bR were examined. The important results are as follows: (t) The removal of Ca2+ from recombinant bR in which charged residues were replaced by neutral ones shifted the retinal absorption to the blue, opposite to that observed in wild-type bR or in recombinant bR in which the H-bonding residue, Tyr-185, was replaced by a non-H-bonding amino acid (Phe). (h) Similar to the observation in wild-type bR, the binding of Ca2+ to the second site gave the observed color change in the recombinant bR samples in which charged residues were replaced by neutral ones. (ii) The residue replacements had no effect on the affinity constants of the four to six weakly bound Ca2+. (iv) The two high-affinity sites exhibited reduced affinity with substitutions; while the extent of the reduction depended on the specific substitution, each site was reduced by the same factor for each of the charged residue substitutions but by different factors for the mutant where Tyr-185 was replaced with Phe(Y185F). The above results suggest that the two Ca2+ ions in the two high-affinity sites are within interaction distance with one another and with the charged residues in the retinal pocket. The results further suggest that, while the interaction between Tyr-185 and the high-affinity Ca2+ ions is relatively short range and specific (with more coupling to the Ca2+ ion in the second affmity site), between the charged residues and Ca2+ ions it seems to be of the electrostatic (e.g., ion-ion) long range, nonspecific type. Although neither Asp-85, Asp-212, nor Arg-82 is individually directly involved in the binding of Ca2+ in these two sites, they might all participate in it. Together with the protonated Schiff base, the charged residues along with Tyr-185 and one or two Ca2+ ions (and probably a few water molecules) seem to form an electrostatically coupled system that is part of a cavity that controls the color and function of bR.Bacteriorhodopsin (bR) is the only protein in the purple membrane of Halobacterium halobium (1, 2). It contains a polypeptide chain of 248 amino acid residues (3), as well as a single retinylidene chromophore bound via a protonated Schiff base (PSB) to the E-NH2 group of Lys-216. Upon absorption of a photon, bR undergoes a photocycle (4) during which the all-trans to 13-cis isomerization of the chromophore takes place followed by the deprotonation of the PSB, and protons are pumped across the cell membrane. The resulting electrochemical proton gradient is then used by the bacteria for some metabolic processes (e.g., ATP synthesis) (4, 5).Well-washed purple membrane patches contain 3-4 mol o...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Effects of genetic replacements of charged and H-bonding residues in the retinal pocket on Ca2+ binding to deionized bacteriorhodopsin.

Zhang

El‐Sayed

Bonet

et al. 1993

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

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Nonaqueous affinity capillary electrophoresis investigation of small molecule molecular recognition

Peddicord

Weber

2002

Electrophoresis

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The conditional binding constants for a bis-guanidinium-like receptor and a series of dicarboxylate ligands have been determined in two buffer/solvent systems, namely 25 mM ammonium acetate/1% acetic acid in acetonitrile/methanol (7:3 v:v) and 30 mM N-methyl morpholine/15 mM methanesulfonic acid in acetonitrile/methanol (9:1 v:v). The latter buffer has not been applied before in capillary electrophoresis. The binding constants in both solvent systems decrease as the dicarboxylate length increases. The binding constants are larger in the less competitive N-methyl morpholine buffer. The dicarboxylates associate only weakly with a dicationic analog of the receptor, p-xylyl trimethylammonium, which is not a hydrogen bond donor.

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The active site of bacteriorhodopsin. Two‐photon spectroscopic evidence for a positively charged chromophore binding site mediated by calcium

et al. 1995

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SYNOPSISThe nature of the chromophore binding site of light-adapted bacteriorhodopsin is analyzed by using all-valence electron MNDO and MNDO-PSDCI molecular orbital theory to interpret previously reported linear and nonlinear optical spectroscopic measurements. A total of 45 binding site models are investigated. The binding site is simulated by including the chromophore, the lysine residue (LYSZl6), the following nearby amino acids ASP,,, ASPIIS, ASPZIZ, THRgo, TRPa6, T R P I~s , TRPlsz, TYR57, TYRs3, and TYRIs5) and zero, one, or two divalent cations. We conclude that the unique two-photon properties of the chromophore are due in part to the electrostatic field associated with a Ca'+ ion near to the chromophore. Four amino acids and three water molecules contribute significantly to the assigned chromophore adjacent calcium binding site (ASPs5, ASP,,,, TYRS7 and TYR,,,), and two conformational minima are predicted. The higher energy conformation has the calcium ion stabilized primarily by ASP,, and the chromophore imine proton by ASP,,,. The lower energy conformation has the calcium ion stabilized primarily by ASPzl2 and the imine proton by ASPs5. The latter configuration is more stable due to strong hydrogen bonding between TYRIs5 and ASP212 coupled with electrostatic stabilization of the divalent cation by TYRS7. Although both tyrosine residues are predicted to exhibit some "unprotonated' character, models involving full deprotonation of either TYR57 or TYRls5 do not fit the spectroscopic data. We conclude that the cation binding site identified in this study is the second high affinity binding site for calcium, and that the chromophore binding site is, to a first approximation, positively charged.The chromophore "lh*t" and "I$*-" states, despite extensive mixing, exhibit significantly different configurational character. The lowest-lying '"&*+" state is dominated by single excitations (> 80% for all models studied) whereas the second-excited "lAg*-'' state is dominated by double excitations (> 70% for all models studied with extensive participation by spin-coupled triplet-triplet excitations).

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Nature of the individual Ca2+ binding sites in Ca2+-regenerated bacteriorhodopsin

Cited by 55 publications

References 27 publications

Effects of genetic replacements of charged and H-bonding residues in the retinal pocket on Ca2+ binding to deionized bacteriorhodopsin.

Effects of genetic replacements of charged and H-bonding residues in the retinal pocket on Ca2+ binding to deionized bacteriorhodopsin.

Nonaqueous affinity capillary electrophoresis investigation of small molecule molecular recognition

The active site of bacteriorhodopsin. Two‐photon spectroscopic evidence for a positively charged chromophore binding site mediated by calcium

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