Photoelectric activity of dried, oriented layers of purple membrane from halobacterium halobium

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 85, No. 1, 1978 November 14, 1978

Pages

PHOTOELECTRIC PURPLE

ACTIVITY

MEMBRANE

OF DRIED,

FROM HALOBACTERIUM K~roly

Institute Academy Received

of Biophysics,

of Sciences,

September

ORIENTED

OF

HALOBIUM

Nagy

Biological

H-6701

LAYERS

383-390

Research

Szeged,

Center,

P.O.Box

521,

Hungarian

Hungary

15,1978

SUMMARY Dried layers of purple m e m b r a n e s c o n t a i n i n g b a c t e r i o r h o d o p s i n o r i e n t e d by an e l e c t r i c field d u r i n g d r y i n g were i n v e s t i g a t e d by p h o t o e l e c t r i c and s p e c t r o s c o p i c methods. There is e v i d e n c e to show that the p h o t o c h e m i c a l cycle of b a c t e r i o r h o d o p s i n in a low h y d r a t i o n state is intact not o n l y s p e c t r o s c o p i c a l l y but also e l e c t r o n i c a l l y in these layers. It has been o b s e r v e d t h a t due to the b l u e - l i g h t excitation of the b l e a c h e d form an interm e d i a t e a b s o r b i n g at 520 nm is enriched. Thus this form seems to occur in the i n h i b i t o r y p a t h w a y of b a c t e r i o r h o d o p s i n . INTRODUCTION Bacteriorhodopsin small p r o t e i n

having

pump

series

of c o n f o r m a t i o n a l

has

protonated /M412/

blue-light

(4),

ground

excitation

- BR system excitation light

as a shunt of the M412

indicate causes

that

(8,9).

This

in the p r o t o n form

and a b s o r p t i o n

form

pump caused

by

(2,6,7).

additional

effect

the

bleached

can be a c c e l e r a t e d

lipid m e m b r a n e -

to g r e e n - l i g h t

in the p h o t o p o t e n t i a l

inhibition

transfer

stable:

on a b i m o l e c u l a r

blue light

a decrease

life-times

and the d e p r o t o n a t e d

of the b l e a c h e d

a

the m o l e c u l e

are r e l a t i v e l y

uptake

measurements

undergoes

the p h o t o c y c l e

two of w h i c h

is a

of a l i g h t - d r i v e n

lead to p r o t o n

with d i f f e r e n t

/BR570/,

halobium

the p r o t e i n

which

During

The proton

Photopotential

green

changes,

state

(2,4).

properties

illumination

intermediates

characteristics

form

Upon

the cell membrane.

several

from H a l o b a c t e r i u m

the unique

proton

through

(1-5).

/BR/

has been

induced

by the

interpreted

by the b l u e - l i g h t

excitation

(10-12).

0006-291X/78/0851-0383501.00/0 383

Copyright © 1978by Academ~ Press, ~c. AHrigh~ ~ reproducnon m any ~rmreserv~.

Vol. 85, No. 1, 1978

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

The p r o t e i n - p i g m e n t /PM/ of H. h a l o b i u m 18). T h e s e

is v e r y

adventages

simple m o d e l

system:

located

stable u n d e r

in the p u r p l e m e m b r a n e extreme

conditions

of B R have b e e n u s e d to c o n s t r u c t dried

electric

field.

response

/up to 500 mV/

parallel

photoelectric

MATERIALS

The

complex

layers

stability

of PM f r a g m e n t s

/for m o n t h s /

of this

a new,

oriented

by a

and the h i g h p h o t o -

system make

and s p e c t r o s c o p i c

(13-

it s u i t a b l e

for

investigations.

AND M E T H O D S

The PM f r a g m e n t s used in the i n v e s t i g a t i o n s w e r e o b t a i n e d by a s t a n d a r d p r o c e d u r e from H. h a l o b i u m strain N R L R I M 1 (19). One drop of a dense P M s u s p e n s i o n in w a t e r was put on a glass p l a t e h a v i n g a c o n d u c t i n g surface. For the o r i e n t a t i o n of the P M fragment a metal e l e c t r o d e w a s p l a c e d above the drop, and a high DC v o l t a g e was a p p l i e d b e t w e e n the c o n d u c t i n g surface of the glass plate and the m e t a l e l e c t r o d e d u r i n g drying. The e l e c t r i c field s t r e n g t h was v a r i e d up to iOOO V/cm, w h e r e the o r i e n t a t i o n / c o n t r o l l e d by m e a s u r i n g the p h o t o r e s p o n s e / was the highest. I l l u m i n a t i o n of the s u s p e n s i o n d u r i n g the d r y i n g p r o m o t e d the o r i e n t a t i o n of the P M fragments. The p h o t o p o t e n t i a l of the d r i e d P M layers was m e a s u r e d w i t h a v i b r a t i n g p l a t e c o n t a c t - p o t e n t i a l m e a s u r i n g e q u i p m e n t (20) /home-made, s e n s i t i v i t y 0.5 mV, r e s p o n s e t i m e O.i s/. The scheme of the set-up is o u t l i n e d in Fig. i. T h e dark s u r f a c e p o t e n t i a l of the l a y e r s was a l w a y s c o m p e n s a t e d . The h u m i d i t y of the layers was kept c o n s t a n t w i t h s a t u r a t e d salt s o l u t i o n s (21). The system was left to e q u i l i b r a t e for 24 h o u r s b e f o r e m e a s u r e m e n t s . The d a t a p r e s e n t e d h e r e were o b t a i n e d w i t h LiCI solution, w h i c h gives 12% r e l a t i v e humidity. D i f f e r e n c e a b s o r p t i o n spectra a f t e r e x c i t a t i o n of the layer w e r e t a k e n p o i n t by p o i n t at 25 nm i n t e r v a l s w i t h a P e r k i n - E l m e r F l u o r e s c e n c e S p e c t r o p h o t o m e t e r M o d e l MPF-3. A f t e r a t w o - m i n u t e e x c i t a t i o n of the PM layers, the e x c i t i n g b e a m was switched off, the m e a s u r i n g b e a m w a s s w i t c h e d on and the change of a b s o r p t i o n of the l a y e r s was m e a s u r e d as a f u n c t i o n of time. A c o n s t a n t r e l a t i v e h u m i d i t y was also m a i n t a i n e d in the p h o t o m e t e r c h a m b e r by the m e t h o d m e n t i n e d above. All the r e s u l t s p r e s e n t e d here w e r e o b t a i n e d on the same sample; s i m i l a r r e s u l t s w e r e o b t a i n e d w i t h a n u m b e r of o t h e r samples. RESULTS

AND DISCUSSIONS

Simple positioning However,

air-drying

of the P M s u s p e n s i o n

of the m e m b r a n e

it is not

sheets o n a g l a s s

a vectorial

a vectorial

proton

pump,

preliminary

condition

causes

orientation

the v e c t o r i a l

384

surface

and,

(22).

as the BR has

orientation

to get a p h o t o r e s p o n s e

a parallel

is the

on PM layers.

An

Vol. 85, No. 1, 1978

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

. . . .

.

:2

! Fig. i. S c h e m a t i c d i a g r a m of surface p o t e n t i a l m e a s u r i n g system used for p h o t o p o t e n t i a l m e a s u r e m e n t s . E: e l e c t r o m e t e r , MC: m o v i n g coil, SC: sensory coil, R: recorder, C: closed chamber, B: vibrating bar, P: p l a t i n u m plate, L: PM layer, G: glass plate w i t h c o n d u c t i n g surface, M: s e m i t r a n s p a r e n t mirror, H: heat filters, FI: green band pass filter /Tmax: 540 nm/, F2: blue band pass filter /Tmax: 405 nm/. Light sources: 200 W m e r c u r y arc lamps /HBO 200, Carl-Zeiss, Jena/.

electric

field o r i e n t s

in the above m e t h o d

the electric

of the PM suspension. layers

were

(17),

form

(17,25).

field

of PM layers

maximum

The

the d i r e c t i o n

was positive.

causes

a small

vectorial

induced

The m a g n i t u d e

on the electric

was

and

with drying dried

field

state

PM

560 nm

/O.D.

could

electric higher

air-drying orientation

be

influenced

field d u r i n g

if the upper

without /small

an e l e c t r i c photopotential/,

of the PM on the glass

by the h y d r o p h i l i c

effect

of the p h o t o p o t e n t i a l the light

385

of

the BR is in d a r k - a d a p t e d

was m u c h

orientation

strength,

around

is in line w i t h the r e s u l t s

of the a p p l i e d

Simple

a preferential

/probably

surface/.

combined

sign of the p h o t o p o t e n t i a l

bu~ the p h o t o p o t e n t i a l

indicating plate

maximum

as in a low h y d r a t i o n

by a l t e r i n g

electrode

field was

(23,24)

In this way v e c t o r i a l l y - o r i e n t e d ,

The a b s o r p t i o n

ref.

drying,

vectorially

obtained.

The a b s o r p t i o n 0.4-0.8/.

the PM f r a g m e n t s

of the glass

on PM layers

intensity

applied

depended during

%'ol. 85, No. 1, 1978

drying,

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the c o n c e n t r a t i o n

The d i f f e r e n c e

observed

different

samples

in sample

preparation

orientation 75%, where response

as d e s c r i b e d

of p h o t o p o t e n t i a l s

above

under

the p h o t o p o t e n t i a l

was

some mY/.

on the dried

intensity

full control.

Samples

/the h i g h e s t

sample

of

parameters

the p h o t o p o t e n t i a l

than half a year.

in w e t air as well

was m e a s u r e d

and the rate of drying.

that not all the relevant

have been b r o u g h t

for m o r e

a green-light

suspension

in the m a g n i t u d e s

indicate

In layers m a d e be r e p r o d u c e d

of the

preserve

relative

could their

humidity

T h e highest

was

photo-

/over P205 / , 500 m Y at

of 18.5 m W / c m 2, w h i c h

decreased

w h e n the

sample was wetted. The m a i n the

same

18,26).

intermediates

in dried P M layers Upon

excitation

in a b s o r p t i o n tion of BR. a change

occurs

in the

of the layers

around

surface

if an additional

green-light

prove

that

totally

blue

/dotted

humidities

intact

of 55-70%

electronically state

shown

the p h o t o p o t e n t i a l

This p a r a m e t e r

in Fig.

the p h o t o p o t e n t i a l simultaneously

(26).

decreases

photoelectric

The results

/regarding

2.

to illuminate

by flash

of BR in these

causes

dependence

2/. The t r a n s i e n t

cycle

the d e p r o t o n a -

excitation presented

layers

the charge

here

is

displacement/

too.

The t i m e - d e p e n d e n c e are

is shown

(16-

an increase

separation

of p h o t o p o t e n t i a l

has b e e n p r o v e d

the p h o t o c h e m i c a l

in low h y d r a t i o n

sities

in Fig.

indicates

by c h a r g e

intensity

of P M

light,

of the PM layers.

light was u s e d

lines

suspensions

which

intensities

of PM m u l t i l a y e r s

at r e l a t i v e

curve

cycle of BR are

by intense

followed

potential

At h i g h g r e e n - l i g h t

activity

412 nm,

and a typical

the e x c i t i n g

the layers

as in aqueous

The d e p r o t o n a t i o n

was m e a s u r e d upon

of the p h o t o c h e m i c a l

in Fig.

of p h o t o p o t e n t i a l s 3. W h e n

decreases

the green

exponentially

386

at two light light with

inten-

is swiched time.

off

The decay

Vol. 85, No. 1, 1978

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

7°

?'

T; a. I

40

20 I00

I00

•E 0

J I ~ - . ~ .

-~

- 4 - -" - 4-

+"~'--

~--.,--

-,

b)

~200

- +-

x - - . x - - x-

-100 ? I

O'-,X)_.-O

100 -200

Green light intensity(mWlcm2) i

®

I

10-2

10"1

I

I

I

I0

0

®

~

8

Time(rain)

12

16

Fi~. 2. D e p e n d e n c e of the p h o t o p o t e n t i a l on the g r e e n - l i g h t intensity V a r i e d w i t h neutral glass filters /continuous curve/. The dotted lines indicate the changes in the p h o t o p o t e n t i a l induced by green e x c i t a t i o n due to the b l u e - 1 ½ g h t excitation. The blue light intensities were: /i/ 0.7 m W / c m , /2/ 2.6 m W / c m 2 and /3/ 13.2 m W / c m ~. Fig. 3. The t i m e - d e p e n d e n c e of the increase and d e c r e a s e of photopote~al following changes in illumination. G and B indicate green and blue light, respectively. The g r e e n - l i g h t i n t e n s i t i e s were: /a/ 9.2 x iO- m W / c m 2 and /b/ 1.8 mW/ cm 2. The b l u e - l i g h t intensity was 13.2 m W / c m 2 in both cases.

can be separated

into three phases w i t h life-times:

T2=60 s and T3=IO0

s. P r a c t i c a l l y

from s p e c t r o s c o p i c

measurements

centration.

Accordingly

charge displacement,

presence

of M412 intermediates.

Difference Fig.

for the d e c r e a s e

as in ref.

/through other

4/. There

is no shoulder

is caused by

(26), i n d i c a t e s the

light

intermediates/

of the M412 con-

which

spectra of PM layers

4. The effect of green b l e a c h i n g

forms BR570 Fig.

absorption

are p r e s e n t e d

is obvious:

to M412

in

it trans-

/curve 1 in

in the s p e c t r u m near 520 nm,

387

s,

the same values were o b t a i n e d

the p h o t o p o t e n t i a l ,

molecular

TI
i.e.

Vol. 85, No. 1, 1978

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S

4

0.05 I E

' 2

0

o,,;/

,.?

3E

,~

/,~ c~...

\

',.), v

O

o-005 I

2 A" P -

',,

II

o\

,

,?

0

c

k%

%1 l

%

l

0e -

u -0.10 %

0"

I

-0.15

Wav¢lcngth(nm)

3bo

4bo

5bo

6bo

7bo

Fig. 4. D i f f e r e n c e a b s o r p t i o n spectra 3.6 s after excitation: curve i: change after g r e e n - l i g h t excitation; curve 2: change c a u s e d by b l u e - l i g h t excitation; curve 3: change after green- p l u s - b l u e - l i g h t excitation; curve 4: d i f f e r e n c e in a b s o r p t i o n of the b l e a c h e d layer caused by blue light /the d i f f e r e n c e b e t w e e n curve 3 and i/.

N520

has not d e v e l o p e d

peak

can be seen around

as shown

in ref.

the results The

(26).

in the a l l - t r a n s

green-light

640 nm,

in ref.

On the other

indicating of N520

(26),

as 0640

cycle of BR, but too/

excitation

excitation

causes

/curve

effect

of g r e e n - p l u s - b l u e

single

green

absorption

appears

is in c o n t r a s t

with

should

light

change,

in the 13-cis

2 in Fig. /curve results

388

4/.

a broad of 0 6 4 0

/in a c c o r d a n c e

a similar

hand,

the a p p e a r a n c e

of this may be that N 5 2 0 m i g h t

observations

Blue-light

time.

The a b s e n c e

presented

interpretation

liminary

at that

but

follow N520. not

follow M412

w i t h other cycle

of BR.

smaller

However,

3/ as c o m p a r e d

pre-

effect

the common to the

in an a b s o r p t i o n

peak

to

VoI. 85, No. 1, 1978

around

BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

520 nm,

i.e.

the c o n c e n t r a t i o n

blue-light

excitation

absorption

spectra

blue

after b l e a c h i n g

light

According process

As blue

transmitted PM,

light

should

be c o n n e c t e d

the i n h i b i t i o n The

with

pathway

results

in the 13-cis of BR,

can regain

cycle,

between

in refs.

by

i.e.

the model

photo-

excitation (2,6,7>,

the p r o t o n

intermediate

the

of the r e g u l a r

the e x t r a c e l l u l a r

it,

the

surface pump

of

of the

is shunted.

of i n h i b i t i o n

N520,

through

which

be closed.

that the

intermediate

and as the M412 excitation

the two

in the

excited

(10-12>,

the d a r k r e a c t i o n

show that

the

must

suggest

the b l u e - l i g h t

connection

here

it was

due to the b l u e - l i g h t

does not reach

presented

by the

also be o b s e r v e d

when

is that m o d i f i c a t i o n

accelerates

so the m o l e c u l e

The r e s u l t s

presented

of BR occurs

proton

could

is e n r i c h e d

(7).

to the model

cycle

N520

of the PM s u s p e n s i o n

of i n h i b i t i o n

chemical M412.

of M412.

of N520

works

N520 m i g h t

in the a l l - t r a n s

of M412 w o u l d m a k e

independent

work

cycles

cycle

a close

of BR isomers.

ACKNOWLEDGEMENTS The author is grateful to Dr. Zs. D a n c s h ~ z y for p r e p a r a t i o n of the p u r p l e m e m b r a n e fragments and to Prof. L. K e s z t h e l y i for helpful discussions. T h a n k s are also due to Mr.K. Frank and Mrs. E. N a g y for their technical assistance. This work was supported by the H u n g a r i a n A c a d e m y of Sciences, UNESCO/UNDP Grant to H u n g a r y No. HUN/71/506/B/OI/13, and by the N a t i o n a l C o m m i t t e e of T e c h n i c a l D e v e l o p m e n t /OMFB/.

REFERENCES i. Oesterhelt, D. and Stoeckenius, W. /1973/ Proc. Natl. Acad. Sci. USA 70, 2853-2857. 2. Oesterhelt, D. and Hess, B. /1973/ Eur. J. Biochem. 37,316-326. 3. Stoeckenius, W. and Lozier, R.H. /1974/ J.Supramol. Struct.2, 269-274. 4. Lozier, R.H., Bogomolni, R.A. and Stoeckenius, W. /1975/ Biophys. J. 15, 955-962. 5. Dencher, N. and Wilms, M. /1975/ Biophys. Struct. M e c h . l , 2 5 9 - 2 7 1 . 6. Hess, B. /1976/ FEBS Lett. 64, 26-28. 7. Hess, B. and Kuschmitz, D. /1977/ FEBS Lett. 74, 20-24. 8. Dancsh~zy, Zs. and Karvaly, B./1976/ FEBS Lett. 72, 136-138. 9. Karvaly, B. and Dancsh~zy, Zs./1977/ FEBS Lett. 76, 36-40.

389

VoI. 85, No. 1, 1978

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

i0. Ormos, P., Dancsh~zy, Zs. and Karvaly, B. /1977] European Seminar on Biological Solar Energy Conversion Systems, Grenoble-Autrans, France, Absr. D-8. ii° Dancsh~zy, Zs., Ormos, P. and Karvaly, B. /1977/ 4th Internat. Congress. on Photosynthesis, Reading, U.K. Abstr. 12. Ormos, P., Dancsh~zy, Zs. and Karvaly, B. /1978/ Biochim. Biophys. Acta 503, 304-315. 13. Mendelson, R. /1976/ Biochim. Biophys. Acta 427, 295-301. 14. Garty, H., Klemperer, G., Eisenbach, M. and Caplan, S.R. /1977/ FEBS Lett. 81, 238-242. 15. Kanner, B.I. and Racker, E. /1975/ Biochem. Biophys. Res. Commun. 64, 1054-1061. 16. Hwang, S.-B., Korenbrot, J.I. and Stoeckenius, W. /1977/ J. Membr. Biol. 36, 115-135. 17. Korenstein, R. and Hess, B. /1977/ FEBS Lett. 82, 7-11. 18. Korenstein, R. and Hess, B. /1977/ Nature 270, 184-186. 19. Oesterhelt, D. and Stoeckenius, W. /1974/ Methods Ensymol. 31, 667-668. 20. Zisman, W.A. /1932/ Rev. Sci. Instrum. 3, 367-8. 21. Weast, R.C. /1970/ Handbook of Chemistry and Physics, 51st edn° E-40, The Chemical Rubber Co. Cleveland, Ohio. 22. Breton, J. /1977/ Thesis, Paris-Sud. 23. Shinar, R., Druck/nan, S., Ottolenghi, M. and Korenstein, R. /1977/ Biophys. J. 19, 1-5. 24. Eisenbach, M., Weissmann, C., Tanny, G. and Caplan, S.R. /1977/ FEBS Lett. 81, 77-80. 25. Kalisky, 0., Goldschmidt, C.R. and Ottolenghi, M. /1977/ Biophys. J. 19, 185-189. 26. Hwang, S.-B., Korenbrot, J.I. and Stoeckenius, W. /1978/ Biochim. Biophys. Acta 509, 3OO-317.

390