Purple membranes (PM) from Halobacterium halobium were incorporated into 7.5% polyacrylamide gels to prevent aggregation which occurs in suspensions at low pH. At pH 7.0, the circular dichroism (CD) spectra and visible absorption spectra of light- and dark-adapted bacteriorhodopsin (bR558, respectively) and the flash photolysis cycle of bR568 in gels were essentially the same as those in PM suspensions. Titration of the gels with hydrochloric acid showed the transition to a form absorbing at 605 nm (bR605 acid) with pK = 2.9 and to a second form absorbing at 565 nm (bR565 acid) with pK = 0.5. Isosbestic points were seen for each transition in both light- and dark-adapted gels. In addition, a third isosbestic point was evident between pH 3.5 and 7. Visible CD spectra of bR568, bR605 acid, and bR565 acid all showed the bilobed pattern typical of bR568 in suspensions of PM. Flash kinetic spectrophotometry (with 40-microseconds time resolution) of bR605 acid and bR565 acid showed transient absorbance changes with at least one transiently blue-shifted form for each and an early red-shifted intermediate for bR565 acid. Chromophore extraction from membrane suspensions yielded all-trans-retinal for bR565 acid and a mixture of 13-cis and trans isomers for bR605 acid.

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We present a location for the retinylidene chromophore in dark-adapted bacteriorhodopsin based on the differences in neutron scattering between purple membrane preparations reconstituted with retinal and with deuterated retinaL The Fourier difference density map contains more peaks than expected, and additional arguments are introduced to exclude artificial peaks caused by the reconstitution techniques or the limited resolution of the diffraction data. The membrane preparation used is necessarily dark-adapted and therefore contains 13-cis-and all-trans-retinal isomers in roughly equal amounts. However, we find only a single position for both isomers. Presumably, the difference in conformation caused by isomerization around the C13-C14 double bond is minimized by rotation around other bonds. The retinal is located between a-helical segments of the rotein and its nearest neighbor (intratrimer) distance is 26 A; the next-nearest neighbor (intertrimer) distance is 38 A.The purple membrane occurs as differentiated patches in the cell membrane of halobacteria. It contains only one protein, bacteriorhodopsin (bR), of molecular weight 26,500; the amino acid sequence is known (1, 2). It has a strong absorption band between 500 and 600 nm due to covalently bound retinal. bR converts light energy into an electrochemical proton gradient, which drives energy-requiring metabolic processes such as ATP synthesis and amino acid transport (3-5). The crystalline array of bR in the plane of the purple membrane has made it possible to determine the membrane structure at high resolution. Henderson and Unwin (6), using low-dose electron microscopy, obtained a three-dimensional scattering density map at 7-A resolution in the plane of the membrane and at 14-A resolution normal to it. The protein apparently consists of seven a-helical rods, about 40 A long and 10 A apart, all oriented roughly perpendicular to the membrane plane and arranged in two parallel rows. This model has been confirmed by Hayward et al. (7). The lipids occupy the interstices of the protein lattice in a bilayer orientation (8).The group of a-helical segments shown below in Fig. 2 Resonance Raman spectra of bR and its photocycle intermediates (17) strongly suggest that the retinylidene Schiff base is directly involved in proton transport across the membrane. This has led us to examine the location of retinal in the membrane. The retinylidene imine can be exchanged if the chromophore is bleached with hydroxylamine under intense illumination, followed by regeneration with added 13-cis-or alltrans-retinal (18,19). In this paper, we report the position of the retinylidene isomers in the membrane plane as determined by a neutron-scattering Fourier difference density map between samples regenerated with normal and with deuterated retinal, respectively. We have reported previously that the f3-ionone ring of the molecule is located near the center of the membrane profile (20). MATERIALS AND METHODSPreparation of Deuterated Membrane. Fully deuterated purple membrane...

Solvent 11. A solution of 2-8 mg of tritiated hydrocarbon in 0.5 ml of CCla was injected into 25 ml of trifluoroacetic acid and 8 aliquots were sealed in 5-ml ampoules with nitrogen flushing. The ampoules were maintained at 70.00 =k 0.06'; after thermal equilibrium was reached, points were removed at intervals and quenched in Dry Ice-acetone. The contents were worked up as above.In the runs with pyrene-2-t the solutions were made up with vacuum line techniques and transferred with argon pressure to tubes which were sealed off with complete exclusion of air. For best results we recommend this type of procedure generally for studies of polycyclic hydrocarbons in trifluoroacetic acid. In several runs using the earlier procedures we noticed varying amounts of decomposition that probably result from radical cations produced by oxygen.The data were handled as first-order kinetics using Perrin's program' or LSKIN~ and were checked for quality with CalComp plots. A typical plot is shown in Figure 2.aniline (pK,. -6.6842). For each kinetic run a 55-ml aliquot of solvent I was syringed into a 100-ml long-necked flask, sealed with a serum cap, and maintained at 25.00 =k 0.02'. A solution of the hydrocarbon in 2 ml of CClr was injected into the flask and shaken vigorously. At intervals, 10-ml aliquots were withdrawn and quenched with excess cold 2 N NaOH. The mixture was extracted with ether or cyclohexane, washed until neutral, and dried. In early runs the evaporated residues were sublimed but this procedure gave irreproducible results; subsequent control experiments showed that sublimation resulted in loss of tritium activity. Hence, the extracts were assayed spectrophotometrically and the samples for counting were prepared by diluting known volumes with scintillation solution.(42) M.Abstract: Various semiempirical MO methods have been applied to the extensive reactivity data now available from protodetritiation of polycyclic aromatic hydrocarbons in trifluoroacetic acid. In common with past experience the simple HMO method is totally inadequate to handle the wide range of structures available. The HMO-w technique, however, is astonishingly satisfactory, undoubtedly because it is an approximation of SCF-a methods. The latter methods are also satisfactory and do not depend in an important way on the specific model or parameter set used. The CND0/2 method also gives a generally excellent correlation. The effect of methyl substituents is well accounted for by CND0/2 although the resulting correlation differs from that of the unsubstituted polycyclic systems. None of the methods gives a satisfactory account of the strained biphenylene system; CND0/2 also is useless in interpreting aromatic substitution reactivities in fluorene and benzocyclobutene.tudies of protodetritiation of polycyclic aromatic S hydrocarbons in trifluoroacetic acid pioneered by Eaborn and his research group2z3 and extended by us4 have provided data for many positions of a number of compounds covering a reactivity range of over six orders of magnitude. Thes...