The titrations of Asp‐85 and of the cation binding residues in bacteriorhodopsin are not coupled

Eliash, Tamar; Ottolenghi, Michael; Sheves, Mordechai

doi:10.1016/s0014-5793(99)00289-6

Cited by 11 publications

(13 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…36 Furthermore, it was shown that the titrations of Asp85 and of the cation binding residues in BR are uncoupled, excluding direct binding of the cation to Asp85. 37 Further supporting the notion that the binding occurs at the membrane surface are solid-state NMR analyses indicating that cation binding effects Ala196, located in the E−F interhelical loop. 38 A study using eosin dye covalently bound to the protein suggested that the cations do not bind to specific sites and bind equally to both membrane surfaces and negatively charged lipids.…”

mentioning

confidence: 72%

Cation Binding to Halorhodopsin

2015

View full text Add to dashboard Cite

A member of the retinal protein family, halorhodopsin, acts as an inward light-driven Cl(-) pump. It was recently demonstrated that the Natronomonas pharaonis halorhodopsin-overproducing mutant strain KM-1 contains, in addition to the retinal chromophore, a lipid soluble chromophore, bacterioruberin, which binds to crevices between adjacent protein subunits. It is established that halorhodopsin has several chloride binding sites, with binding site I, located in the retinal protonated Schiff base vicinity, affecting retinal absorption. However, it remained unclear whether cations also bind to this protein. Our electron paramagnetic resonance spectroscopy examination of cation binding to the halorhodopsin mutant KM-1 reveals that divalent cations like Mn(2+) and Ca(2+) bind to the protein. Halorhodopsin has a high affinity for Mn(2+) ions, which bind initially to several strong binding sites and then to binding sites that exhibit positive cooperativity. The binding behavior is pH-dependent, and its strength is influenced by the nature of counterions. Furthermore, the binding strength of Mn(2+) ions decreases upon removal of the retinal chromophore from the protein or following bacterioruberin oxidation. Our results also indicate that Mn(2+) ions, as well as Cl(-) ions, first occupy binding sites other than site I. The observed synergetic effect between cation and anion binding suggests that while Cl(-) anions bind to halorhodopsin at low concentrations, the occupancy of site I requires a high concentration.

show abstract

mentioning

confidence: 72%

Cation Binding to Halorhodopsin

2015

View full text Add to dashboard Cite

show abstract

“…Both of these proposals leave the Asp-85 group protonated in the blue membrane and, therefore, block the proton acceptor in the photocycle but by different means. Another study has shown that the titrations of the Asp-85 and the color-controlling site are not coupled (43). It has become a difficult task to assign whether there is a cation in the interior of the protein and, thus, to explain the exact role of the cation in the proton-pumping mechanism.…”

Section: Discussionmentioning

confidence: 99%

The Role of the Native Lipids and Lattice Structure in Bacteriorhodopsin Protein Conformation and Stability as Studied by Temperature-dependent Fourier Transform-Infrared Spectroscopy

Heyes

El‐Sayed

2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

We report the effect of partial delipidation and monomerization on the protein conformational changes of bacteriorhodopsin (bR) as a function of temperature. Removal of up to 75% of the lipids is known to have the lattice structure of the purple membrane, albeit as a smaller unit cell, whereas treatment by Triton monomerizes bR into micelles. The effects of these modifications on the protein secondary structure is analyzed by monitoring the protein amide I and amide II bands in the Fourier transform-infrared (FT-IR) spectra. It is found that removal of the first 75% of the lipids has only a slight effect on the secondary structure at physiological temperature, whereas monomerizing bR into micelles alters the secondary structure considerably. Upon heating, the bR monomer is found to have a very low thermal stability compared with the native bR with its melting point reduced from 97 to 65°C, and the premelting transition in which the protein changes conformation in native bR at 80°C could not be observed. Also, the N-H to N-D exchange of the amide II band is effectively complete at room temperature, suggesting that there are no hydrophobic regions that are protected from the aqueous medium, possibly explaining the low thermal stability of the monomer. On the other hand, 75% delipidated bR has its melting temperature close to that of the native bR and does have a pre-melting transition, although the pre-melting transition occurs at significantly higher temperature than that of the native bR (91°C compared with 80°C) and is still reversible. Furthermore, we have also observed that the reversibility of this pre-melting transition of both native and partially delipidated bR is time-dependent and becomes irreversible upon holding at 91°C between 10 and 30 min. These results are discussed in terms of the lipid and lattice contribution to the protein thermal stability of native bR.

show abstract

“…An older ESR study found 5 Mn 2ϩ binding sites with fairly high affinities (Dunach et al, 1987). It was also postulated that the titration of Asp85 and the color-controlling site are not coupled (Fu et al, 1997;Eliash et al, 1999). The differences observed in the high-affinity sites for Ca 2ϩ and Mn 2ϩ suggest that their binding to bR are not identical, and may regulate the color differently.…”

Section: Binding Sites Of the Different Cations To Bbrmentioning

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

View full text Add to dashboard Cite

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.

show abstract

The titrations of Asp‐85 and of the cation binding residues in bacteriorhodopsin are not coupled

Cited by 11 publications

References 45 publications

Cation Binding to Halorhodopsin

Cation Binding to Halorhodopsin

The Role of the Native Lipids and Lattice Structure in Bacteriorhodopsin Protein Conformation and Stability as Studied by Temperature-dependent Fourier Transform-Infrared Spectroscopy

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

Contact Info

Product

Resources

About