Spectroscopic study of photoreceptor membrane incorporated into a multilamellar film

Vol.

92. No.

February

4, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

27, 1980

Pages

1266-1272

SPECTROSCOPIC STUDY OF PHOTORECEPTOR MEMBRANE INCORPORATED INTO A MULTILAMELLAR K.J.

Qothschild

N.A.

R. Sanches Department MA 02215,

of Physics *Department

Received

December

Clark*,

and T.L.

FILM

K.M.

Rosen

Hsiao

and Department of Physiology Boston University, Boston, of Physics, University of Colorado, Boulder, CO go309 17,1979

SUMMARY: A new method of orjenting membrane fragments is described which has ~_-been used to produce multilamellar films of photoreceptor membrane. These films display linear dichroism which indicates the membranes are well oriented. Fourier transform infrared spectroscopy and far ultraviolet circular dtchroism both indicate that the visual transducing membrane protein rhodopsin contains extensive alpha-helical structure which is oriented prodominantly perpendicular to the membrane plane. INTRODUCTION: ------

iJe report

incorporate

biological

extensively

studied

method

study

(A study visual

transducing

protein visible

by circular

to determine

membrane rhodopsin

film

the

multilayer

specific

formed surface

native

from induced

bovine

elsewhere

rod outer

Our segments.

Rhodopsin,

changes,

when applied

relative

(h-9)

are extremely

membrane

information

MATERIALS -------.

This

of membrane

spectroscopy

of specific same methods

(2-5).

(l).)

conformational

about

the

to

to the

(h) , has been extensively

methods

small

and alpha-helices

order

developed

of the membrane.

from

absorption these

(1)

similar lipids

structure

membrane

orientatfon the

films

reported

is

Although

that

of a method

membrane

and can reveal

can reveal

chromophore

the

of this

(9).

we show here film

optimal

and infrared

dichroism structure

contrast,

into

photoreceptor

membrane

application

arrays

destroying

involves

by ultraviolet.

unable

not

of purple

rhodopsin

membranes

to promote

while

initial

on the

multilamellar

is designed

fragment3

here

groups.

the studied

as

well

sensitive they

as to

are

In

to photoreceptor orientation

to the membrane

of the plane.

AND fjlm formation was accomplished using the .--- __ Multilayer --- METHODS: This developed spin-dry isopotential centrifugation technique (1). method consists of ultracentrifuging suspended membrane fragments onto a substrate surface which is normal to the centrifugal force, while simultaneouslv evaporatlng the solvent. A specially desIgned isopotential recently

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92, No.

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centrifugation cell (ICC) is used which fits into the bucket of an SW25.2 The ICC allows for the utilization of any substrate swinging bucket rotor. which can bend to a 12 cm radius of curvature without cracking. In practice thin glass, quartz flats, polyethylene, aluminum foil, mica and some IR A bucket cap with transmitting materials such as KRS-5 and AgCl can be used. a small lo-50 micron hole allows controlled evaporation of the suspending solvent into the centrifuge vacuum. Bovine rod outer segments (ROS) with a 280 nm/500 nm absorption ratI. of approximately 2 are isolated using a sucrose density gradi.ent method (10). The ROS are osmotically lysed and then washed 3 times in distilled water. Electron microscopy shms this treatment results in vesicles approximately 1 micron in diameter (10). One ml suspensions of these vesicles with an OD ranging from .01-l are spun at 11,OOOxg in an ICC for 17 hrs at 4'C. Optically uniform films with a thickness of .5 to 50 microns are obtained. Films can also be prepared from solutions containing a 5-fold molar excess of 11-cis retinal. These films when mounted in a cuvette containing 50 mM phosphate buffer, pH 6.8, or distilled water exhibit full regenerability as determined by recovery of the 500 nm absorption subsequent to a bleaching flash (11). Multilamellar orientation of the photoreceptor membrane was checked by (i) microscopic examination which reveals a strong linear dichroism indicating the t1.0

i

0.9

F 0.8

0.6

I

300

400

500

WAVELENGTH

600

(nm)

Figure 1. Visible-ultraviolet absorption spectrum of dark-adapted photoreceptor membrane film after equilibration for 30 minutes approximately 75X relative humidity. Peaks are found at 5001~~ Film was formed using the isopotential spin-dry method from a .5 OD ROS suspension which had been washed 3 times In distilled thickness as measured by light microscopy was approximately 25 measurements were made on a Gary 219 UV-VIS spectrometer. The ratio was estimated after visually correcting for the scattering

1267

at and 275 nm. 1 ml solution HF. Film microns. All 280

nm/SOO

background.

of

nm

Vol.

92, No.

4. 1980

BIOCHEMICAL

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polarization of the retinylidene chromophore is predominantly in the film plane (12) , (ii) linear dichroism of the 500 nm rhodopsin absorption band (cf. shows the rhodopsin chromophore tilt relative CO the film plane Fig. 1) , which is 17 degrees in agreement with microspectrophotometric measurements on a single rod outer segment (13) . These results indicate that the photoreceptor membrane film consists of a multilayer array. A lffaering of the 280 nm/500 nm absorption ratio from 2 for solubilized rhodopsin to approximately 1.4 for the film can also be accounted for by the linear dichroism of the retinylidene chromophore. A similar effect, for example, has been observed in rhodopsin gelatin films (14) . RESULTS

AND DISCUSSION

photoreceptor peaks

are

membrane due, peptide

unusually

high closer

rhodopsin

Fig.2 film

to (8)

.5

, as

is well

the

(7 ,8 ,15,16)

II/amide

i.nfrared cm

amide .

One

I absorption

found as

the

900-1800

to

groups amide

shms from

respectively,

rhodopsin

value

:

for for

suspensions most

proteins

-1

absorption .

I and

The

(17)

S, ROS,

cm

-1

11 modes

of

ratio, of

1657

amide

feature

spectrum

these of

.X.

disoriented

of

and of

1545

a cm

the

peaks

is

In

contrast dried

an , an films

.

1.23,

I ii a

I 0.81-

0.601

T 1800

~7

IT1600

U(cm-I) Figure 2. Infrared absorption spectrum of a bovine photoreceptor membrane film, recorded at room temperature at normal incidence. The measurement was made in a dual beam Fourier transform IR spectrometer (Digilab FTS-14, Cambridge. MA). The spectrum was obtained at 4 cm-l resolution with 100 scans of the sample and 50 scans of the reference beam. The sample was prepared on a AgClwindow using the isopotential spin-dry method. It had an optical density at 500 nm of 0.3 OD. The ratio S of the amide II/amide I band intensities was calculated using the peak maximum as the intensity of the band, and taking the broken line as the base line. The sample was exposed to light at - 30X humidity, resulting in a Meta I like absorption at 480 nm. for films unexposed to light. Almost identical spectra with S-. 8 were found

1268

-1

S of

Vol.

92,

No.

BIOCHEMICAL

4, 1980

Recently

it

,

membrane

fi.lms

is

alpha-hel<.ces

is

predicted

to

at

beam.

of

43

measurements (11) to

led

an

rhodopsin actual

Figure angle

Q (n

bottom

curve

plotted

of

3.

he

is

was s =

alpha-helix

of to

as tilt

change

membrane A peaks which

be even

of S, the amide II/amide related to ~0 through Snell’s law) of the family) to 0.9 (in intervals (for a derivation cf. ref. 11) :

purple

to

the

test

of

a vertically consistent an

average

Linear

normal. also

with

led

to

exhibited

a simjlar lower

(cf.

cross

mosaic Fig.

in

conformation lcxrer

(11).

I ratio, on the sample tilt for pa. ranging from 0 (the of 0.1). Ihe equation

pU is the u-helix order parameter, p,-<(3cos ‘0,-l) /2> angle of the o-helix axis relative to the membrane normal. that the membranes were perfectly oriented in the film. The correspond to experimental values obtained for an unbleached optical density at 500 nm of 0.1 OD and thickness approximately Ihe points fall close to the curve corresponding to pg0.30, cross is the value obtained using the ifpectrum shown In Fig. value (0.8) we have p,,=O.45 and B,X -37 .

1269

and Oa is the It was assumed squares

film

with an 5 microns.

ecx -43’.

2.

For

3). (20)

p,(3sin2a-I)+1 P,~(*~x-

where

for

variation

having:

non-alpha-helical

will

order

is

alpha-helices

37 degrees

40%

In

(19).

to

the

of

bacteriorhodopsfn

C.i,j relative

5 microns,

dependence

45 x 0.49 0.69

of

this

COMMUNICATIONS

S value

measured

11 and

I,

approximately

much

hi.Sh

we

3,

Fjg.

than

of as

fj.lm,

a group

amide

thicker

estimate

in

relative

the

the

plane

angles

S for

RESEARCH

orientation membrane

shcktn

degrees

contains

average

the

that

membrane

As

Samples

.

(18)

different

of

BIOPHYSICAL

preferential to

variation

dichroism

spread,

the

tjlted

tilt

estimate

demonstrated

photoreceptor

incident

upper-limit

the

in

film

polarized

Since

due

effect

the

the

been

perpendicular

a similar S as

has

AND

The

that

s

Vol.

92, No.

4, 1980

The film

far

also

BIOCHEMICAL

ultraviolet

supports

alpha-helices.

the As

solutions

of

immersed

in

would

a CD effect

helical has

in

Fig.

(9)

is

H20.

alpha-helices the

shown

distilled to

to

existence

rhodopsin

perpendicular

due

circular

the

Since

dichroism

spectrum

of

predominantly

4,

the in this

its and been

nm Moffit

the

film is

(21-23)

n-n

of

light

proposed

in

the purple

detergent dry

or

incident

arrangement

remaining of

oriented

found

partially for

The

membrane rhodopsin

band

active , the

for

photoreceptor

either

* transitions

reported

COMMUNICATIONS

perpendicular

disappearance.

the

RESEARCH

210

band

axis

explain

recently

BIOPHYSICAL

absent

alpha-helix

bands

AND

of

activity amide

may

(21-24).

membrane

be Such

films

(2%. In multilayer

conclusion, films

the enables

incorporation

of

photoreceptor

structural

information

to

membrane be

obtained

into about

15

-10 I

1

90

210

WAVE

230

LENGTH

250

(nm)

Figure 4. Far ultraviolet circular dichroic spectra of photoreceptor membranes. The solid line is the spectrum of rhodopsin in 1% digitonin solution and the broken line shws the spectrum of isopotential spin-dried photoreceptor membrane film. These measurements were made on a Csry 61 The beam was incident normal to the membrane spectropolarimeter at 23’C. was 30 seconds and plane. The dynode voltage was less than 0.4 kv, the period Very thin films (OD at 500 nm
1270

Vol.

92, No.

rhodopsin.

measurements the

segment

magnetically

membrane

diamagnetism

Since systems

containing

films

This

specific

membrane

is

segments

(27)

generally

proteins,

is

applicable reticulum additional

COMMUNICATIONS

and ultraviolet

circular

photoreceptor

membrane

alpha-heltces

in agreement

The detection

such as sarcoplasmic

RESEARCH

to unorlented of rhodopsin

(26).

is

BIOPHYSICAL

of the tnfrared

relative

result

rod outer

our method

AND

change

orientation

plane.

oriented

findings.

of

preferential

to the membrane outer

the

In particular,

dichroism reflect

BIOCHEMICAL

4. 1980

with

the

perpendicular existence

of IR dichroism

in

also

with

consistent to other

our

biological

and reconstituted studies

of rod

should

membranes be possible.

We wish to thank W. DeGrip, J. Korenbrot and P. Bran for AcknaJledgements: ---~.-- ---helpful discussions and V. Culbertson for technical assi.stance. This work was supported by a grant from the NIH-NET (KJR) and FAPESP & IFQSC-USP, Brazil FourSer transform infrared spectroscopy Gas done at the (W . Polarized Material ScCence Center, ?lIT, Cambrtdge, ?!A.

REFERENCES 1. Clark,N.A., Rothschild,K.J., Si.rn0n.B.A. and Luippold,D.A. (1979) (submttted to Biophys. J.) 2. Levine,Y.K., Bailey,A.I., and Wilkins,M.H.F. (1968) Nature 220, 577-578. 3. Akutsu,H., Kyogoku,Y., Nakahara,H., and Fukuda,K. (1975) Chem. and Phys. of Lipids 12, 222-242. 4. Caber,B.P., Yager,P., and Peticolas W.L. (1978) Blophys. J. 22, 191-207. 5. Griffin,R., Powers,L. and Pershan,P.S. (1978) Biochem. 17, 2718-2722. 6. Ebrey,T.C. and Hontg,B. (1977) Quart. Rev. of Biophys. R_, 129-184. 7. Osborne,H.B. and Nabedryk-Vi.ala,E. (1977) FEBS Letters 85, 217-220. 8. Rothschild,K.J., DeGrip,W., and Sanches,R. Biochim. Biophys. hcta (in press). 9. Rafferty,C.N., Cassim,J.Y., and McConnel1,D.C. (1977) Biophys. Struct. Plechanism2, 277-320. 10. DeCrip,W.J., Daemen,F.J.Y., and Bonting,S.L. (1979) in Methods In Enzymology. Vitamins and Coenzymes (IlcCormick,D.B. and Wright,L.D. eds.) Academic Press, New York (in press). 11. Rothschild, K.J., Rosen,K.%., Clark, N.A., Sanches, R. and Hsiao, T.L. (submitted to Biophysical J.) 12. Abrahamson,E.W., Japar,S.N. (1972) The Structure, spectra, and reactivity of visual pigments. Handbook of Sensory Physiology. Vol. VII/l Photochemistry. Edited by Dartnal1,H.J .A. Berlin, Springer-Verlag. 13. Lfebman,P.A. (1969) Annals of New York Acad. Sci. 13, 250-264. 14. Wright,W.E., Brcwn,P.K. and Wald,C.J. (1972) Gen. Physiol. 59, 201-212. 15. Fraser,R.D.B and HacRae, T.P (1973) in Conformations in Fibrous Proteins and Related Synthetic Polypeptides, New York, &ademic Press. 16. Susi, H. (1969) in Structure and Stability of biological Macromolecules (S.N. Timasheff and C.D. Fasman, eds.), New York, Dekker, pp 575. 17. Blout,E.R., de Loze,C., and AsarJourian,A. (1961) J. Am. Chem. Sot. 83, 1895-1900. 1R. Rothschild,K.J. and Clark,N.A. (1979) Biophys. J. 25, 473-487.

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Henderson,R. and Unwin,P.N.T. (1975) Nature 257, 28-32. Litman,B.J. (1979) Photochem. Photobiol. 29, 671-677. M0ffitt.W. (1956) J. Chem. Phys. 25, 467-478. Tinoco Jr,I. (1964) J. Am. &em. Sot. 86, 297-298. Mandel,R. and Holzwarth,G. (1972) J. Chem. Phys. 57, 3469-3477. Woody, R.W. and Tinoco J. I., (1967) J. Chem. Phys. 46, 4927-4945. M~cci0,D.D. and Cassim,J.Y. (1979) Biophys. J. 22, 427-440. Chabre,M. (1978) Proc. Natl. Acad. Sci. USA 7_5, 5471-5474. Michel-Villaz.M., Saibil,H. and Chabre.M. (1979) Proc. Natl. Acad. 7_6_, 4405-4408.

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