&ion

Rex. Vol. I5 pp. 1081-IOY6. Pergamon Press 1975. Prtnted in Great Britain.

THE ISOMERIC CONFIGURATION OF THE BACTERIORHODOPSIN CHROMOPHORE LU_Y YEH JAN Division of Biology, California Institute of Technology, Pasadena. California 91125. U.S.A. (Receiced

13 Yocemher

1974; in reaised form

19 January

1973)

Abstract-Oesterhelt and Stoeckenius in 1971 found a pigment in Halobacterium halohium which they called bacteriorhodopsin because it resembles rhodopsin in many aspects. The isomeric configurations of its chromophore were studied by thin layer chromatography of the retinal and of the retinal oxime derivatives. The dark form of bacteriorhodopsin (R& contains 13-cis retinal. in contrast to the 1I-cis retinal of rhodopsin. Both the RJ7n and the M,,2 forms of bacteriorhodopsin were found to contain all-rrans retinal. Implications of the different isomeric forms found in bacteriorhodopsin are discussed.

Bacteriorhodopsin’ was discovered in Halobacterium ltolohittru by Oesterhelt and Stoeckenius (1971). The striking similarities between the visual pigment of animals, rhodopsin and bacteriorhodopsin include light sensitivity, the presence of retina1 as the chromophore, the Schiffs base linkage between retina1 and a lysine residue, the location of the pigment in-a membrane and its close packing, the hydrophobic nature of the protein, and the sequences of the photochemical reactions (Blaurock and Stoeckenius, 1971; Oesterhelt and Hess, 1973; Oesterhelt and Stoeckenius, 1973a; Stoeckenius, persona1 communication). When bacteriorhodopsin is kept in the dark or under dim red light, it absorbs maximally at 560 nm (R,,,). This will be called the dark-form of bacteriorhodopsin. At room temperature, light converts R5d0 to a form absorbing maximally at 570~1 (R&, which reverts slowly to R560 in the dark with a halflife of 20min at 35°C (Oesterhelt and Stoeckenius, 1971; Oesterhelt, Meentzen and Schuhmann, 1973) (Table la). R5,e is also light sensitive. Studies performed at various low temperatures revealed three intermediates (K6e5, Lsso and M4r2) in the photochemical reactions of RS7e, kinetically resembling prelumi-, lumi- and metarhodopsin (Stoeckenius, 1974, personal communication; Morton, 1972) (Table lb). The MJ12 form reverts to RS,e with a half-life of 5 msec at room temperature (Oesterhelt and Stoeckenius, 1973a). The conversion of Mzll to Rs7,, can be slowed down either by keeping the temperature below -40°C or by introducing ether to the purple membrane suspension in high salt solution (Stoeckenius, 1974, personal communication). Indeed, we found that at room temperature the dark-form, RS6,,, in ether-saturated high salt solution is converted by light to M4r2. and that in the dark M4r2 “reverts” to R5,o with a half-life of about 13sec, as reported by Oesterhelt and Hess (1973). We also confirmed

the results of Oesterhelt and Hess (1973) that RS7e in ether-saturated high salt solution is light-sensitive and is bleached to Mill by light (Lipson and Jan, unpublished results) (Table lc). The isomeric configuration of retinal in bacteriorhodopsin has been studied with purple membranes suspended in low salt solution and in dimethylsulfoxide solutions by Oesterhelt et al. (1973). The present report concerns the isomeric configuration of the bacteriorhodopsin chromophore in the dark-form (R&, the Rj70 form and the M.,r: form.

MATERIALS .i_-XD.METHODS Materials

The R,L, strain of Halobacterium halohium (Oesterhelt and Stoeckenius, 1973b) was used. Pelleted cells were resuspended in minimal volume (about l/30 of the culture Table 1 (a) Bacteriorhodopsin

RjeO g R,,, r = 20minat 3% (b) Bacteriorhodopsin

in low salt solution

R 570 *

K605

> -M;C

> -I-WC

i M *I1 ->_so.c

I Ls50

(c)Bacteriorhodopsininether-saturatedhighsalt hr Lo r 4

’ AS the Editor has pointed out, rhodopsin means “to appear rosecolored” from the Greek. Bacteriorhodopsin has its absorption maximum at 56Onm and is better called an iodopsin. The author fully agrees with the Editor. However, the old terminology is used here because it has been used in numerous publications in the past 3 yr.

in low salt solution

-

solution*

hv M +,z

4

R570

13 xc at room temp.

* Light flashes of duration 30 set are used in this experi-

ment. Photochemical reactions of Rs6e after very short flashes of light are currently under study and the detailed mechanism is much more complex than w&t is indicated here (Stoeckenius. personal communication). 1081

volume) of basal j;llr. mized uith s:o,sk solutions oi I mg ml D&X and RNase to u~bt: a rinal concentration of about 5OO/lg DSase and RNass per liter culxrs. homogenized in a glass homogemzer. dialyzed eurensivsly against a larse volume of 0.05 &ITris-HCl. pH 7.75, first a[ room temperature ior 2 hr and then at -I C overnight. Purple membrane was spun dou-n in an SS-3-l rotor at 13.COOrpm for 30 min. resuspended in a minimal vol of distilled Hater. and further purl&d on a linear sucrose gradient (XX-50 per cent) (Oesrerhelt r*rai., 1973). 1l-15 retinal was a nift of Hoffman-LaRoche. Inc.. NutIcy. New Jersey. All-&~ retinal and I3-cis retinal were purchased from Sigma Chemical Co.. St. Louis. Missouri. Silica gel chromatogram sheets (10 y 20cm without Huorescent indicator) were obtained from Eastman Kodak Co., Rochester. New York. Petroleum ether (b.p. 2WO’C) was a product of J. T. Baker Co.. Phillipsburg. N. J. 2-methyl-2hapten-&one was purchased from K. Sr K. Laboratory. Cleveland, Ohio.

I I-cis ntfd 13-15sretinnls and retinal

Srpnrario,l o/‘&-trans. oxirws

Five i. of 2-3 mg, ml I I-c&. I3-cis and all-rrotls retinal in 9jO/, ethanol were applied on rhe same silica gel sheet with the retinal samples as standards. For retinal oxime standards. 25 i. of I l-cis. I3-cis or all-rrans retinal in 95:,{ ethanol was reacted with 25 L of 2 ZI hydroxylamine in SOS; ethanol at room temperature for 30 min. The reaction mixtures were evaporated under nitrogen, then resuspended in petroleum ether (b.p. X-WC). One-third or two-thirds of each petroleum ether solution of retinal oxime was applied at each spot as standards. The appbcation of samples to silica gel sheets and the development of thin layer chromatograms, as well as the preparation of samples, were performed under dim red light. Separation of retinal isomers was achieved by developing silica gel sheets at -I’C in petroleum ether (b.p. 2s 4O’Ctacetone (100:3; v, v) (solvent .A)(Akhtan. Blosse and Dewhurst, 1968). Separation of different isomers of retinal oxime was performed by developing silica gel plates at 4’C in petroleum ether (b.p. 2@-4b’C) -2-meth$2-hapten6-one (11:2. v/v) (solvent B) (Oesterhelt et n/.. 1973: Planta. Schwiiter. ‘Chdiard-dit-Jean, Ruegg, Kofler aid Isler. 1962). Retinals were detected by their yellow color on silica

(a)

I

To obtain chromophore from the dark-form of bacteriorhodopsin. 12 ml of purple membrane f - 1mg bacterior-

u(al

( b)

8

I

00

Oll-?KJ~,

E.xtracrion of chrornophow fPom rhr dark-$x-m of bnctrriorhodopsin awl possible artifacrs inrrotllrcrti chrouyh thermnl isomerixtion durirg prcpnrcrtiou

-cm

(b)

0

For the separation of isomers of rerlnal. silica gel sheets ttere developed in solLent .A three tlrn

The isomeric configuration of the bacteriorhodopsin chromophore.

&ion Rex. Vol. I5 pp. 1081-IOY6. Pergamon Press 1975. Prtnted in Great Britain. THE ISOMERIC CONFIGURATION OF THE BACTERIORHODOPSIN CHROMOPHORE LU_Y...
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