Fi g. 1. Ti me -r es ol ve d X- ra y di ff ra ct io n pa tt er n of pu rp le me mb ra ne ( pH 9, 10 ° C) be fo re an d af te r xenon flash lamp excitation. Bragg peaks of a magnitude as great as 7 Å are observed, utilizing a time resolution of 244 ms. BR was illuminated at the beginning of the 11th frame. Bacteriorhodopsin ( BR ) is a proton transporting me mb ra ne pr ot ei n, dr iv en by th e ene rg y of ab so rb ed photons. During the first step of proton transport, the absorption of a photon by retinal, located inside B R , i n d u c e s a c o n f o r m a t i o n a l c h a n g e i n t h e molecule from an all-trans to a 13-cis configuration. A s e q u e n c e o f r e a c t i o n s f o l l o w s t o f a c i l i t a t e t h e tr an sp or t of pr ot on s by BR . Se ve ra l in te rm ed ia te s t a t e s h a v e b e e n c h a r a c t e r i z e d b y d i f f e r e n c e spectra in the visible range as J, K, L, M, N, and O. R e p r o t o n a t i o n o f t h e r e t i n a l S c h i f f b a s e i s a n i n t e g r a l r e a c t i o n i n B R p r o t o n p u m p f u n c t i o n ; t h e S c h i f f b a s e r e c e i v e s a p r o t o n f r o m D 9 6 d u r i n g t h e M - N t r a n s i t i o n i n B R p h o t o c y c l e . B e c a u s e t h e p K o f D 9 6 , l o c a t e d w i t h i n a h y d r o p h o b i c microenvironment, is unusually increased, D96 has a protonated form in the unphotolyzed state. Upon the release of the proton, the pK of D96 is lowered by an additional reaction. Therefore, a critical event enabling the lowering of the D96 pK must occur at the M-N transition. TIME-RESOLVED X-RAY DIFFRACTION EXPERIMENTS OF PURPLE MEMBRANE 50 100 150 0 10 20 30 40 0 6000 4000 2000 0 CCD pixels Time frames (244 ms /frame) 2000 4000 6000 0 To study the structural transition from the M to the N inter media te, we perfo rmed time- resol ved X- r a y d i f f r a c t i o n o f p u r p l e m e m b r a n e a t t h e S A X S s t a t i o n o f B L 4 5 X U ( F i g . 1 ) [ 1 ] . S t a c k e d p u r p l e m e m b r a n e s ( t w o - d i m e n s i o n a l c r y s t a l s o f B R ) p l a c e d o n a m y l a r s h e e t , r e l a x e d a t p H 9 a n d e x c i t e d a t 1 0 ° C b y a x e n o n f l a s h , i m m e d i a t e l y f o l l o w i n g t h e s t a r t o f d i f f r a c t i o n a c q u i s i t i o n s . B y exposing to X-rays, perpendicular to the membrane pl an e, st ac ke d pu rp le me mb ra ne s di ff ra ct X- ra ys circularly in a manner similar to powder diffraction. D i f f r a c t e d i m a g e s w e r e a v e r a g e d c i r c u l a r l y . T o decompose the component in this reaction, the da ta w e r e a n a l y z e d b y s i n g u l a r v a l u e d e c o m p o s i t i o n ( SV D ) ( Fi g. 2 an d Fi g. 3 ). Th re e co mp on en ts ar e dist ingu isha ble from the SVD anal ysis . U spec tra give eithe r the diffr actio n profi les or the diffe rence p r o f i l e s o f i n d e p e n d e n t c o m p o n e n t s ; V s p e c t r a display the time course. U1 is similar to the original diffraction pattern of the purple membrane, except for its negative sign, which is influenced by the sign Fig. 3. V spectra of wild-type BR ( pH9, 10 ° C) o b t a i n e d b y S V D a n a l y s i s f o r w e i g h t e d di ff ra ct io n da ta . V sp ec tr a in di ca te th e ti me course of the corresponding U spectra. Fig. 2. The U spectra of wild-type BR ( pH9, 10 ° C) obt ain ed by SVD ana lys is for wei ght ed dif fra cti on d a t a . T h i s f i g u r e d i s p l a y s t h e t h r e e m a j o r components with the singular values. U spectra are e i t h e r t h e d i f f r a c t i o n p r o f i l e s o r t h e d i f f e r e n c e profiles of independent components. 1.0 0.5 0.0 -0.5 -1.0 -0.5 0.0 -0.4 -0.3 -0.2 -0.1 S2 =666 0.14 0.12 0.10 0.08 0.06 0.04 S3 =59 S1 =253835 U2 U3 S (Å - 1 ) U1 o f V 1 . V 1 d e m o n s t r a t e s l i t t l e t i m e d e p e n d e n c e , indicating that U1 does not change throughout the photocycle. Thus, the first component is composed primarily of the time-independent, basic structure of B R . V 2 a n d V 3 , h o w e v e r , c h a n g e d r a m a t i c a l l y during the flash excitation at time 0, indicating that two unique intermediate conformations exist in the d e c a y p r o c e s s o f w i l d - t y p e B R f o l l o w i n g f l a s h excitation. As the M and N intermediates only were detected spectrophotometrically in this time period, t h e t w o i n t e r m e d i a t e c o n f o r m a t i o n s c a n b e a s s i g n e d a s t h e s e i n t e r m e d i a t e s . T h e t h r e e components, derived from the SVD analysis, were r e c o n s t i t u t e d f r o m g l o b a l f i t a n a l y s i s t o c r e a t e d i f f r a c t i o n p r o f i l e s o f t h e t h r e e s t a t e s . A b s o r p t i o n a n d F T I R m e a s u r e m e n t s i n d i c a t e t h a t t h e reconstituted profiles are likely to correspond to the u n p h o t o l y z e d s t a t e , t h e a b s o r p t i o n d i f f e r e n c e b e t w e e n N a n d t h e u n p h o t o l y z e d s t a t e a n d t h e difference between M and the unphotolyzed state, -0.4 -0.2 0.0 V3 40 30 20 10 0 Time (s) -0.3 -0.2 -0.1 0.0 V2 -0.0711 -0.0710 -0.0709 -0.0708 -0.0707 V1 re sp ec ti ve ly . Ut il iz in g di ff er en ce Fo ur ie r an al ys is , we examined the two difference diffraction profiles t o i d e n t i f y t h e d o m a i n s u n d e r g o i n g l a r g e conf orma tion al chan ges ( Fig. 4 ). The proj ecti ons, m a p p i n g o n t o t h e m e m b r a n e p l a n e o f t h e t w o components, demonstrate changes characteristic to the M and N intermediates, respectively. The slow component contains changes near F and G helices; t h e p o s i t i v e p e a k o n t h e o u t s i d e o f t h e F h e l i x b e c o m e s h i g h e r t h a n t h e p o s i t i v e p e a k o f t h e G h e l i x , a c h a r a c t e r i s t i c p r o p e r t y o f t h e N i n t e r m e d i a t e . Th e an al ys is of th e fa st co mp on en t re ve al ed th at t h e p o s i t i v e p e a k o n t h e o u t s i d e o f t h e F h e l i x reaches a position as high as the positive peak of the G helix, indicating the F helix alters its structure in this manner during the M-N transition. A l t h o u g h t h e r e i s d i s a g r e e m e n t c o n c e r n i n g structural changes during the M-N transition [2] , our r e s e a r c h r e v e a l s t h a t B R c h a n g e s i t s s t r u c t u r e Fig. 4. Difference Fourier maps projected onto the membrane plane of purple membrane samples (pH9, 10 ° C). (a) Difference map of the N intermediate and the unphotolyzed state. (b) Difference map of the M intermediate and the unphotolyzed state. The dashed line indicates the outline of the BR trimer. Capital letters indicate the rough positions of the seven BR helices. References [ 1] T. Oka, N. Yagi, T. Fujisawa, H. Kamikubo, F. Tokunaga and M. Kataoka, Proc. Natl. Acad. Sci. - in press. [2] S. Subramaniam et al. , J . Mol. Biol. 287 ( 1999) 145; H. Kamikubo et al. , Biochemistry 36 (1997) 12282. [3] M. Nakasako et al., FEBS Lett. 292 (1991) 73. Toshihiko Oka SPring-8 / RIKEN E-mail: oka@ spring8.or.jp ( a ( b p r i m a r i l y i n t h e M i n t e r m e d i a t e [ 3 ] . O u r d a t a d e m o n s t r a t e s t h a t g l o b a l s t r u c t u r a l c h a n g e s a l s o o c c u r d u r i n g t h e M - N t r a n s i t i o n . T h e o b s e r v e d structural change at the F helix likely increases the a c c e s s i b i l i t y o f t h e S c h i f f b a s e a n d D 9 6 t o t h e cytoplasmic surface, facilitating the proton transfer reactions beginning with the decay of the M state. W e c o n c l u d e t h a t a l a r g e , t w o - s t e p s t r u c t u r a l c h a n g e is integral in the proton pumping activity of BR.
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