Effect of ionic environment on the protein fluorescence of bacteriorhodopsin

A C T A U N I V E R S I T A T I S L O D Z I E N S I S FOLIA BIOCHIMICA ET BIOPHYSICA 2 , 1983

E r z s e b e t B a l i n t , B e l a K a r v a l y , Z o l t á n V arkonyi EFFECT OF IONIC ENVIRONMENT ON THE PROTEIN FLUORESCENCE

OF BACTERIORHODOPSIN

The p r o t e i n f l u o r e s c e n c e s p e c t r a and p o l a r i z a t i o n o f b a c t e r i o r h o d o p a i n e x c i t e d a t 293 nm and a t room te m p e ra tu r e was i n v e s t i g a t e d i n s o l u t i o n c o n t a i n i n g c h l o r i n e - , a c e t a t e - and r h o d a n i d e - i o n s aB q ue nc h ers o f f l u o r e s c e n o e i n S or en se n and B r i t to n - R o b i n s o n b u f f e r s i n th e pH-range from 4 to 10. T hese q u en ch e rs d i d n ot a f f e c t th e f l u o r e s c e n o e and p o l a r i -z a t i o n s p e c t r a b u t c o n s i d e r a b l e e f f e o t o f the c o m p o si t io n o f b u f f e r s was fou nd e s p e c i a l l y on the f l u o r e s c e n c e p o l a -r i z a t i o n s p eo t -ru m . The c h a n g es i n d i c a t e t h a t some o f the b u f f e r c o n s t i t u e n t s ( c i t r i c a c i d , a c e t i c a c i d , p h osp ha te i o n s ) may e x e r t an e f f e c t on th e t e r t i a r y s t r u c t u r e o f b a c - t e r i o r h o d o p a i n i n a d d i t i o n to th e d i r e o t p H - e f f e c t .

B a c t e r i o r h o d o p a i n (BR) i a a p u r p l e p r o t e i n - p i g m e n t complex i n th e c e l l membrane o f s e v e r a l h a l o b a c t e r i a [ 1 1 , 12'], which forms th e m o l eo u l a r b a s i s o f a new ty p e o f p h o t o p h o s p h o r y l a t i o n [ 1 4 ] . Upon i l l u m i n a t i o n BR u n d e r g o e s a r a t h e r c o m p l i c a t e d ph otoch emi-c a l c y c l e w ith a number o f p h o t o m e t r i c a l l y d e f i n e d s t a t e s [ 1 8 ] , I t works a im u lt a n e o u a ly a s a l i g h t d r i v e n proton-pump [ 1 7 ] , a p h o t o e l e c t r i o g e n e r a t o r o f c u r r e n t [ 1 ] , and a d e l i c a t e r e g u l a t o r o f th e l i g h t - i n d u c e d membrane p o t e n t i a l [ 6 ] , A l l t h e s e f u n c t i o n s a r e c o n n e c t ed among o t h e r s w i th c o n f o r m a ti o n a l c h a n g e s i n the p r o t e i n m o ie t y . S in c e th e c o n f o r m a t i o n a l chang e o f th e p r o t e i n i a i n i t i a t e d by l i g h t - q u a n t a a b a o r b e d by th e r e t i n a l chromophore, an ene rg y excha nge between the r e t i n a l and the p r o t e i n moiety s h o u l d be assum ed [ 4 ] . A tte m p ts t o r e v e a l the r o u t e s and mecha-n ism o f t h i s e mecha-n e r g y - e x c h amecha-n g e p r o c e s s have r e c e n t l y been made, by m ea s u ri n g c e r t a i n f l u o r e a c e n c e c h a r a c t e r i s t i c s o f th e r e t i n a l pig ment complexed w i th th e p r o t e i n [1 3» 7 ] .

th e r o l e o f the t ry p to p h a n r e s i d u e s i n th e m o le ou l ar meohanisms o f d i f f e r e n t f u n c t i o n s o f BR [1 5, 1 0 ] . In t h i s r e p o r t , i n or d er t o m o n ito r the s t a t e s and i n t e r a c t i o n s o f a ro m a t i c amino a c i d r e -s i d u e -s , e s p e c i a l l y t h o s e o f t r y p t o p h a n s , p r o t e i n f l u o r e s o e n c e p r o p e r t i e s o f BR were i n v e s t i g a t e d i n s o l u t i o n s c o n t a i n i n g C l " , CN“ , SCN” i o n s a s f l u o r e s c e n o e q u en c h er s a t d i f f e r e n t p H -v a lu e s .

M a t e r i a l s and methods

= ■■ ■ '' ' " "!.. ^

P u rp l e membrane p a t c h e s c o n t a i n i n g BR were i s o l a t e d from H a l o b a c t e r i u m h al ob iu m s t r a i n N-jM-| by s t a n d a r d p r o c e d u r e s o f e x t r a c t i o n and p u r i f i c a t i o n [ 1 6]• The membrane p a t o h e s were s u s -pend ed i n S o re n se n or B r i t t o n - R o b i n s o n b u f f e r s a t p H -va lue s b etween 4 and 10 and c o n t a i n i n g q u e n c h ers (KSGN, KCIO^, CH^COOK) i n 0 . 2 5 , 0 . 5 and 1 M c o n c e n t r a t i o n . The BR c o n c e n t r a t i o n s o f th e s a m p l e s were a d j u s t e d to an o p t i c a l d e n s i t y o f 0 . 3 a t 285 nm.

The a b s o r p t i o n s p e o t r a were m easured w i th a UNICAM SP 1800 r e c o r d i n g s p e c t r o p h o t o m e t e r . The f l u o r e s c e n c e s p e c t r a and po-l a r i z a t i o n d e g r e e s were r e c o r d e d w ith a PERKIN-LLMER MPF-44A s p e - c t r o - f l u o r i m e t e r u s i n g 1 cm s t o p p e r e d q u a r tz c e l l s . The h a l f - - w i d t h s o f e x c i t i n g and o b s e r v a t i o n b an ds were 5 nm. The s p e c -t r a were c o r r e c -t e d f o r -th e s p e c -t r a l -t r a n s m i s s i o n o f -th e e m i ss io n monochromator and f o r th e s p e c t r a l s e n s i t i v i t y o f th e d e t e c t o r . C o r r e c t i o n s o f f l u o r e s o e n c e s p e c t r a f o r r e a b s o r p t i o n were a l s o made; s e c o n d ar y f l u o r e s c e n c e e f f e c t s were n e g l i g i b l e . The i n f l u -enc e o f l i g h t - s c a t t e r i n g c o u l d n o t be e l i m i n a t e d , no s e l e c t i v e s c a t t e r i n g oc c u r ed which c o u l d have f a l s i f i e d th e s p e c t r a l ch a-r a c t e a-r i s t i c s m ea s u a-re d . A l l m easua-rem ents wea-re made on s a m p l es o f th e same BR e x t r a o t , a t room t e m p e r a t u r e .

R e s u l t s and d i s c u s s i o n

The r e l a t i v e a b s o r p t i o n s p ec tr u m o f n a t i v e BR h a s two main b an d s c e n t e r e d a t 2 80 nm (w it h two s h o u l d e r s ) , and w it h maximum a t 567 nm ( F i g . 1 ) . A s m a l l , p r a c t i c a l l y c o n s t a n t a b s o r p t i o n w i t h i n the 3 0 0 - 4 5 0 nm r e g i o n i s p ro b a b ly due t o l i g h t - s c a t t e r i n g .

F i g . 1. R e l a t i v e a b s o r p t i o n s p ec tr u m o f BR i n S o re n s e n b u f f e r pH = 7

Widmo ró ż ni c ow e p o c h ł a n i a n i a b a k te ri o r o d o p s y n y (BR) w b u f o r z e S o r e n s e n a , pH = 7

OTHOCHTeJibHbiti cneKTp norJioneHHH OaKTepHopononcHHa (BP)

b Óy$epe CapeHceHa, pH =* 7

The u l t r a v i o l e t band c o n s i s t s m a in ly , o f c o n t r i b u t i o n s from t y r o s i n e and t r y p to p h a n . The e x a o t number o f t r y p to p h a n s and ty -r o s i n e s i n th e complex f o -r m a t i o n i s n o t known ( s e e e . g . [ 8 ] ) . C o n s i d e r i n g th e s p e c t r a l p r o p e r t i e s o f p h e n y l a l a n i n e , t y r o -s i n e and try p to p h a n F i g . 1 i n d i c a t e s t h a t o s c i l l a t o r s a b s o r b in g a t 29 0 run a re p r i m a r i l y e l e c t r o n i c t r a n s i t i o n s o f t r y p to p h a n , r a t h e r th a n th o s e o f th e o th e r two a r o m a t i c amino a c i d r e s i d u e s .

The f l u o r e s c e n c e e m i s s io n sp ec t ru m d ep end s upon th e wavelength o f e x c i t a t i o n ( F i g . 2 ) . At 265 nm e x c i t a t i o n th e e m i s s i o n s p e c -trum d i s p l a y s a maximum a t 300 nm and a s h o u l d e r a t 312 nm. For 280 nm e x c i t a t i o n e m i s s i o n w i th maximum'305 nm and a s h o u l d e r a t 314 nm i s c h a r a c t e r i s t i c , w h ile w it h 2 93 nm the e m i s s i o n maxi-mum i s l o c a t e d a t 311 nm and a s h o u l d e r a p p e a r s a t 320 nm. The f l u o r e s c e n c e s p e c t r a a r e s h i f t e d to w a rd s s h o r t e r waves a s compa-r e d t o th e u s u a l p r o t e i n f l u o r e s c e n c e . Very s l i g h t i n f l u e n c e o f i o n s u s u a l l y f u n c t i o n i n g a s quenchers c o u l d be o b s e r v e d on th e e m i s s i o n p r o p e r t i e s even a t h i g h (a b o u t 1 m o l a r ) c o n c e n t r a t i o n . pH a f f e c t e d the p r o t e i n f l u o r e s c e n c e , e s p e c i a l l y i t s p o l a r i -z a t i o n p r o p e r t i e s ( F i g . 3 ) . These s p e c t r a have minima i n the

F i g . 2 . R e l a t i v e f l u o r e s c e n c e s p e c t r a o f BR i n S o re n s e n b u f f e r pH = 7 , a t d i f f e r e n t e x c i t a t i o n w a v e l en g t h s

Widma ró ż n ic o w e f l u o r e s c e n c j i BR w b u f o r z e S ó r e n s e n a , pH = 7 , d l a róż n yc h d ł u g o ś c i f a l ś w i a t ł a w z b u d z a j ą c eg o

Ot h o cHTejibHbie cn eK T pti (¿uiyopecueHuKH BP b 6 y $ e p e O ap eH ceH a , pH =* 7 , n p a p a 3 Jn m H u x AJiHHax b o ji h B03 6yiKnaio[qero C B e i a . d e g r e e o f p o l a r i z a t i o n a bo ve 310 nm and maxima i n th e 3 70 -3 9 0 nm r e g i o n . The p o l a r i z a t i o n s p e c t r a e x h i b i t f i n e s t r u c t u r e a s w e l l . S c a t t e r i n g e x e r t s i t s i n f l u e n c e p r i m a r i l y n ea r th e e x o i t a t i o n w a v e le n g t h . S o r e n s e n and B r i t t o n R o b i n s o n b u f f e r s do n ot i n f l u e n c e a p p r e -c i a b l y th e a b s o r p t i o n i n the u l t r a v i o l e t bu t a l t e r th e p o l a r i -z a t i o n p a t t e r n ( F i g . 4 ) . On the b a s i s o f th e a b s o r p t i o n and f l u o r e s c e n c e e m i s s io n s p e c t r a i n th e u l t r a v i o l e t r e g i o n , one may a ttem p t t o i n d e n t i f y th e a b s o r b i n g and e m i t t i n g c e n t e r s .

The u l t r a v i o l e t a b s o r p t i o n band c o n s i s t s m ainly o f c o n t r i b u -t i o n s from -tr y p -to p h a n and -t y r o s i n e . T y r o s i n e , which a b s o r b s i n p a r t i n th e same s p e c t r a l r e g i o n a s tr y p t o p h a n , e m i ts a s i n g l e band w ith maximum a t 303 nm i n aqueous s o l u t i o n s , but i t s f l u o r e -s c e n c e i s g e n e r a l l y quenched by t ry p to p h a n i f th e l a t t e r i s p r e s e n t i n th e p r o t e i n [ 9 ] . I t i s more p l a u s i b l e t o assume an a n o m a l ou s ly b l u e s h l f t e d t r y p t o p h a n - f l u o r e s c e n c e th an an u n u s u a l t y r o s i l f l u o r e s c e n c e which d o m i na te s th e r o o m -t em p er a t u re p ro-t e i n f l u o r e s c e n c e o f n a ro-t i v e BR. In p r o t e i n s which o o n t a i n t r y p

-Sôrensen buffer

30

^ = 2 9 3 ™

\\

pH*4 —

\\

pH=7 —•

20

\\\ pHsK)--.

k i " " 10 XV.

A(nm)

1 6 Ô ’ 4ÔÔ" P i g . 3 . P o l a r i z a t i o n a p e o t r a o f p r o t e i n f l u o r e s c e n c e o f BR a t d i f f e r e n t pH. B u f f e r c o m p o s i t i o n : pH = 4 : 0 .0 5 5 M CgHoOy, 0 .2 7 5 M NaOH, 0 . 0 4 5 M HC1 ; pH = 7 : 0 .0 2 7 M KH2 P0, 0 .0 4 0 M Na2H P 0 ,; pH = 10:

0 . 0 6 5 M C2H5ON, 0 .0 6 5 M NaOl, 0 .0 3 5 M NaOH

Widma f l u o r e a c e n c j i b i a łk o w e j BR w ró ż n y c h w a r t o ś c i a o h pH. S k ła d b uforów : pH= 4: 0 , 0 5 5 M C6Hft0 7 , 0 ,2 7 5 M NaOH, 0 ,0 4 5 M HC1; pH = 7: 0 , 0 2 7 M KH2P 0 ,, 0 ,0 4 0 M Na2HP0A; pH = 10: 0 , 0 6 5 M C7HcON, 0 ,0 6 5 M

Na C l , 0 ,0 3 5 M NaOH

CneKipu Ó ejiK O B O ił <J>JiyopecijeHaHH BP b p a 3 Ji H q H H X pH. CociaB t f y ^ e p o B :

pH = 4 : 0 ,0 5 5 M CgHgOy, 0 ,2 7 5 M NaOH, 0 ,0 4 5 M HC1; pH = 7s 0,0 27 M KH ,P0 ,, 0 ,0 4 0 M Na,HP0,5 pH = 10: 0 ,0 6 5 M G,HR0N, 0 ,0 6 5 M N aCl,

0,0 35 M NaOH

to p h a n , th e e m i s s i o n s p e c t ru m i s th ou g h t to be a s t r o n g f u n c t io n o f th e environment o f th e t ry p t o p h a n s [ 2 ] . E i a i n g e r and N a v o n [ 3 ] have shown t h a t i n n o n - p o l a r environm en t th e emis-s i o n maximum moveemis-s by a b o u t 1 0-20 nm to w a rd emis-s emis-s h o r t e r w a v e emis-s. T h is s h i f t c an be e x p l a i n e d by the p r e s e n c e o f t r y p to p h a n r e s i d u e s s i t u a t e d i n a l o w - p o l a r i t y e nv iron m en t, and i n t e r a c t i n g w ith t h e i r s u r r o u n d i n g s th rou g h t h e i r im i n o g r o u p s v i a h yd rog en b ond s. Such c o n d i t i o n s may be e a s i l y a t t a i n e d i n th e s a c k form ed by the p r o t e i n i n which th e p ol y en e c h a in o f t h e r e t i n a l chromophore i s n e s t e d , and the p r o t e i n s a r e n ot a c c e s s i b l e to s i m p l e quencher i o n a even i f they a r e p r e s e n t i n h ig h c o n c e n t r a t i o n . pH seems t o e f f e c t H-b ond s which a r e n ot d i r e c t l y i n v o l v e d i n th e f o r m a t io n o f th e p u r p l e c om p lex , a t l e a s t i n th e pH rang e 4 1 0 . The s t r u c -t u r e may be c o n s i d e r a b l y a f f e o -t e d by -th e c o m p o s i -ti o n o f -th e buffer even i f th e pH i s k e p t c o n s t a n t .

«Sûrensen buffer

1 0

Brlttgr^obinson

A(nm) ---►

3ÔÔ P i g . 4 . P o l a r i z a t i o n s p e c t r a o f p r o t e i n f l u o r e s c e n o e o f BR i n d i f f e r e n t b u f f e r s . B u f f e r c o m p o s i t i o n . S o r e n s e n : 0 . 0 2 7 M KH2 P0<, 0 .0 4 0 M Na2HP04 ; B r i t t o n - R o b i n s o n : 0 .0 2 6 M C2H / 0 , 0 . 0 2 6 M H-,BrO/ 0 .0 2 6 M H3P04 , 0 .0 6 9 M NaOH Widma p o l a r y z a c j i f l u o r e s c e n c j i b i a łk o w e j BR w ró ż n y c h b u f o r a c h . S k ła d b uforów : b u f o r S ó r e n s e n a : 0 , 0 2 7 M KH2 P 0 , , 0 ,0 4 0 M Na2H P0 ,; b u f o r B r i t t o n a - R o b i n s o n a : 0 ,0 2 6 M C7H ,0 , 0 , 0 2 6 M H^BrO/ 0 ,0 2 6 M

H3P04 , 0 ,0 6 9 M NaOH

CneKTpH noJiHp03ai;nH 6ejiK0B0it (fiJiyopecueHUHH BP b pasjiHMHUx Oytjiepax.

CocTaB 6y$epoB . Ey$ep CapeHceHa: 0 ,0 27 M KH2P 0 , , 0 ,0 4 0 M N a2HP04 ; Ey$ep BpHTTOHa-PoCHHcoHa: 0 ,0 26 M 02H /0 , 0 , 0 2 6 M H3B r 0 4, 0 ,0 2 6 U

H3P04, 0 , 0 6 9 M NaOH

With th e u s e o f P 1 a t t ’ s n o t a t i o n s [19 ] the p r o b a b l e c l a s s i f i c a t i o n s o f th e r e l e v e n t a b s o r p t i o n and e m i s s i o n t r a n s i -t i o n s o f -t r y p -t o p h a n , we p r o p o se -t h a -t -th e t r y p t o p h a n s o f BR e x i s t i n a t e a s t two d i f f e r e n t s t a t e s , one o f them r e f l e c t s th e p r e -s e n c e o f an ex tre m e ly l o w - p e r m i t t i v i t y m ic ro e n vir on m e n t, and th e s t r u c t u r e o f th e e m i s s i o n s p e c t r a i s i n p a r t due t o th e t w o - l e - v e l f l u o r e s c e n c e o f t ry p t o p h a n [ 5]. T h e i r p o l a r i z a t i o n p rop er tie s a r e d i f f e r e n t and o f o p p o s i t e s i g n . Our f i n a l c o n c l u s i o n i s th at th e r e t i n a l chromophore o f b a o t e r i o r h o d o p s i n e x i s t s i n an extrem - l y h i d r o p h o b i c environm ent which i s l i n e d by f u n c t i o n a l l y imp o r t a n t tr y imp to imp h a n r e s i d u e s . I t i s imp o s s i b l e , t h a t e x c i t a t i o n r e -d i s t r i b u t i o n t a k e s p l a c e w i t h i n e x c i t e -d t r y p t o p h a n s .

Ao knowledgement 3

Thanks a r e due t o Z. Danoshazy f o r p r e p a r i n g th e p u r p l e mem-b ra n e and t o P r o f . L . S z a l a y f o r h e l p f u l d i a c u a a i o n a . REFERENCES [ 1 ] D r a c h e v L.

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[1 7] O e a t e r h e l t D ., Aquew. Chemio. I n t l . Edn. 1 5 , 17 ( 1 9 7 6 ) . [18 ] 0 r m 0 b P . , D a n c a h a z y Z. , K a r v a l y B . , B io c h im . e t B i o p h y a . A ota 5 0 3 , 304 ( 1 9 7 8 ) . [1 9] P l a t t J . R . , I . Chem. Ph ya.

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