Isomeric Composition of Retinal Chromophore in Dark-Adapted Bacteriorhodopsin1

Maeda, Akio; Iwasa, Takeshi; Yoshizawa, Tôru

doi:10.1093/oxfordjournals.jbchem.a131855

Cited by 76 publications

(63 citation statements)

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“…The Schiff base bond of the retinylidene chromophore is easily formed under physiological conditions and incubation of opsin with the retinal that has the appropriate configuration results in the formation of pigment [14][15][16][17]. The thioester bond is difficult to obtain under the mild conditions at which protein is not denatured.…”

Section: Discussionmentioning

confidence: 99%

Reconstitution photoactive yellow protein from apoprotein and p‐coumaric acid derivatives

et al. 1995

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We report reconstitution of photoactive yellow protein (PYP) from apoPYP and p-coumaric acid derivatives. The addition ofp-coumaric acid to the apoPYP sample did not result in the recovery of PYP. In contrast, yellow products were obtained by the addition of p-coumaryl thiophenyl ester or pcoumaric anhydride to the apoPYP sample, the absorption spectra of which were indistinguishable from the spectrum of intact PYP. Our findings provide strong evidence that PYP has the p-coumaryl chromophore. This reconstitution technique opens the way for further biophysical studies of PYP using artificial chromophore analogs.Key words'." Photoactive yellow protein; Photoreceptor protein; p-Coumaric acid; Chromophore; Reconstitution; Ectothiorhodospira halophila 1, IntroductionPhotoactive yellow protein (PYP) is present in Ectothiorhodospira halophila as a soluble small chromoprotein [1]. It has a broad absorption band, the absorption maximum of which is at 446 nm [1]. It is considered a photoreceptor protein for the negative phototaxis of E. halophila [2]. PYP has a photoreaction cycle comprised of several intermediates, the spectral and kinetic properties of which are very similar to those of the retinal proteins in halobacteria [3 5]. Formation of the bathochromic photoproduct occurs upon light absorption, the photoproduct being converted to a near-UV intermediate in a microsecond time scale and reverting to the ground state in a subsecond time scale. These intermediates correspond to the K (batho) or L (lumi) and M (meta) intermediates of the retinal proteins. The relation between PYP and the retinal proteins, therefore, is a matter of interest, but the nature of the PYP protein moiety is very different from the moieties of the retinal proteins [6,7]. Retinal proteins are membrane proteins composed of seven transmembrane helices [8,9] and have molecular weights of 24,000-40,000. PYP is a soluble small protein of 14 kDa which crystal structure analysis to 1.4-A resolution has shown to have the ot/fl-fold structure [7].The PYP chromophore first was thought to be retinal because its photochemical properties are similar to those of retinal proteins [10]. The chromophore-binding site was tentatively assigned to the lysine residue at the 111 position spectroscopy has shown that the molecular weight of the chromophore is 147, smaller than that of retinal [6]. Moreover, the chromophore binding site is a unique cysteine residue at position 69 [6]. These findings suggest that the chromophore is not retinal. Recent studies have shown that the PYP chromophore is p-coumaric acid (4-hydroxycinnamic acid) which binds to the cysteine residue via a thioester bond [11,12] (Fig. la,b).Although studies based on NMR analysis of the chromophore released from digested PYP [11] or high-resolution crystallography of PYP [12] clearly have shown that PYP has the p-coumaryl chromophore, direct confirmation would depend on the reconstitution of PYP from its apoprotein and pcoumaric acid. Moreover, an effective reconstitution technique would pr...

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Section: Discussionmentioning

confidence: 99%

Reconstitution photoactive yellow protein from apoprotein and p‐coumaric acid derivatives

et al. 1995

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“…All-trans-Retinal was purchased from Sigma Chemical Co. 13-cisRetinal was isolated from a mixture of isomers produced by irradiation of all-trans-retinal and purified using high performance liquid chromatography [14].…”

Section: Retinal and 13-cis-retinalmentioning

confidence: 99%

Reason for the lack of light‐dark adaptation inpharaonis phoborhodopsin: reconstitution with 13‐cis‐retinal

1995

FEBS Letters

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The reconstitution of pharaonis phoborhodopsin was performed by incubation of its opsin with 13-cis-retinal. Spectrum change was very slow, and two phases of the change were observed: the first and second phases are due to the transient formation of 13-cis pigment and spontaneous isomerization to all-transretinal, respectively. Slow binding supports an idea that the retinal binding pocket of ppR is highly restricted. Being bent in the configuration, 13-cis-retinal cannot be accommodated in the pocket due to the steric hindrance. This is a possible reason for the lack of light-dark adaptation.

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“…2). In the case of BR in the purple membrane, BRL has only all-trans retinal as its chromophore but BRD has both all-trans and 13-cis retinals in equimolar amount (15,16). The present experiment showed that the ratios of 13-cis to all-trans retinals in T-BRL and T-BRD were 13.0 2 2187.0 2 2 and 51.3 t 3148.7 -C 3, respectively.…”

Section: Discussionmentioning

confidence: 43%

“…In the case of irradiation of BRD in purple membrane at -190°C, two kinds of batho-intermediates, namely bathotrans-BR and batho-13-cis-BR, were formed (14), because BRD in purple membrane is an equimolar mixture of trans-BR and 13-cis-BR (15,16). The same was true in the T-BRD system, because the hplc analysis of retinal isomers extracted from T-BRD revealed that T-BRD was a mixture of T-BR' (5 1.3 ?…”

Section: Cooling Effrcts On Spectra Of T-brd and T-brl; Appearance Ofmentioning

confidence: 99%

Photochemical reactions of bacteriorhodopsin in Triton X-100 solution studied by low temperature spectrophotometry

Iwasa¹,

Tokunaga²,

Yoshizawa³

1985

Can. J. Chem.

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The photochemical reaction of purple membrane solubilized with Triton X-100 (T-BR) was investigated by low temperature spectrophotometry. The batho- and meta-intermediates of T-BR were observed to resemble bacteriorhodopsin in native purple membrane. Two photoproducts characteristic of the T-BR system were found, which were named the "490-nm complex" and the "380-nm complex". The 490-nm complex was in thermal equilibrium with T-BR in the dark. Cooling T-BR to low temperature favoured the 490-nm complex, which was photoinsensitive. On the other hand, the 380-nm complex was produced by warming the batho-intermediate and reverted to the original T-BR. The meta-intermediate of T-BR may possibly be in thermal equilibrium with the 380-nm complex. On the basis of the above results, the possible role of the membrane structure was discussed

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Isomeric Composition of Retinal Chromophore in Dark-Adapted Bacteriorhodopsin1

Cited by 76 publications

References 0 publications

Reconstitution photoactive yellow protein from apoprotein and p‐coumaric acid derivatives

Reconstitution photoactive yellow protein from apoprotein and p‐coumaric acid derivatives

Reason for the lack of light‐dark adaptation inpharaonis phoborhodopsin: reconstitution with 13‐cis‐retinal

Photochemical reactions of bacteriorhodopsin in Triton X-100 solution studied by low temperature spectrophotometry

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