Vol. 67, No. 3, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ANATURALCDLABELTOPROBETHESTRUCTUREOFTHEPURPLE MEMBRANE

FROM

COUPLING

EFFECTS.

M. P. Heyn’: ” Dept.

P. -J.

HALOBACTERIUM

Bauer’

of Biophysical

+ Institut

and N A.

Chemistry, KFA,

fur Neurobiologie,

Received

September

HALOBIUM

Dencher

BY MEANS

OF EXCITON

’

Biozentrum,

4056

Basel,

517 Jiilich

1, Fed.

Rep.

Switzerland Germany

23,1975

SUMMARY Coupling between retinal chromophores on adjacent bacteriorhodopsin molecules in the hexagonal surface lattice of the purple membrane from Halobacterium halobium RI leads to exciton circular dichroism (CD) spectra in the 567 nm absorption band. Uncoupling by solubilization of bacteriorhodopsin in Triton X-100 results in a loss of this couplet. In dimethylsulfoxide (DMSO)/water mixtures, both exciton peaks disappear and a positive CD band develops at 460 nm. The change is reversible. It is suggested that DMSO increases the protein mobility in the membrane. Bleaching causes the concerted disappearance of both exciton peaks around 567 nm, and the appearance of optical activity in the 412 nm band. Because of its strong dependence on geometry, this CD effect appears to be a sensitive probe to study changes in protein mobility and in protein-protein interactions. INTRODUCTION Both

rhodopsin

valently

and bacteriorhodopsin

bound

dopsin

to the protein.

in suspensions

of a single (1).

positive

In contrast

highly

mobile

(2),

are

immobilized

The

close

necessary retinal

the occurrence

of neighboring

description

of the CD spectra

mentioning

possible

CD signal

hexagonal mobility

exciton

evidence may

that

such

be used

was

coupling

at the absorption

maximum

to study

ple membrane. 897

molecules

in the purple lattice

coupling

the protein

are

membrane (3,4,

5, 6, 7).

molecules effects

are between

molecules. suspensions

recently

effects

of rhoconsists

bacteriorhodopsin

interactions,

co-

as in detergents

of the protein

membrane

of retinal

the CD spectrum

surface

of exciton

of purple

molecule

the rhodopsin

molecules

a regular

for

chromophores

natural

in which

and the restricted

conditions

strong

as well

the bacteriorhodopsin

packing

region

approximately

membranes,

and form

present

membranes

centered

to disc

a single

In the visible

of disc band

contain

occur

A brief in water,

presented and show

arrangement

(8).

We

how

this

in the pur-

Vol. 67, No. 3, 1975

MATERIALS

BIOCHEMICAL

AND

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

METHODS

Suspensions of purple membranes were prepared from Halobacterium halobium RI following the standard method (9). CD measurements were carried *with a Cary 61. To study bleaching effects, the cell compartment and cell holder assembly were modified to allow illumination in a direction perpendicular to that of the measuring beam. A Schott KL 150 lamp with a light conductor system was used for this purpose. Tests with various substances showed that with these modifications CD spectra could be recorded under photostationary conditions. RESULTS

AND

CD spectra

DISCUSSION

of purple

of purple

membrane

700 nm.

Note

with

crossover

feature

the protein

long

part band

wavelength

chromophores (2) a combination There

at 574 nm.

(567 nm),

and an exciton tive

the asymmetry

We will

with

on adjacent of two for

at least

positive

to the band

a positive which

short

bands

the discussion are

band

two

centered

for

wavelength

lobe

is due to the interaction

different the second

molecules

transitions

with

interpretation

WAVELENGTH,

from

opposite

250 to

of opposite to this

possible

sign

unique explana-

at the absorption

of the retinyl

observed

bacteriorhodopsin

the CD spectra

pH 6.88

of the two

restrict there

1 shows

buffer

is due to the interaction

(analogous

lobe,

is no evidence

in amplitude

of a broad which

Fig.

in phosphate

for which

(A) a superposition

maximum

suspensions.

suspensions

of the spectrum,

tions:

with

membrane

rhodopsin

residue at 500nm),

and an equal between

nega-

retinal

in the membrane; circular

dichroism.

in the absorption

spectrum.

nm

Fig. 1. The CD spectrum of a light adapted suspension of purple membranes from H. halobium in 0. 025 M phosphate buffer (pH 6. 88). Molar ellipticities in this and other figures are based on spectrophotometrically determined Temperature 20° C. chromophore concentrations.

898

BIOCHEMICAL

Vol. 67, No. 3, 1975

Our

experiments

Since

on the other

the crossover

absorption ly with posed

to this

that

pretation

also

and that

shifts

be reflected

we observed absorption

in equal

indeed

mately

equal

equal

reduction

which

is approkimately

tisfied.

in amplitude

effects rigid

the square

changes

shifts

in both

peaks

time

of both

for

of the extinction

symmetrical-

amplitude

a simple

peaks

coefficient

CD amplitudes maximum

(lo),

adaptation

by approxi-

are

in an

65 nm apart,

dimer

model.

have

of the bacteriorhodopsin

exciton

light

8 nm as in the

of conditions

from

interbe equal

resulted

coupled

estimated

This

maximum

increased

two

and for

should

Upon

of about

peaks

a number

distances,

the pro-

567 nm.

lobes.

redshift

The

at the

in amplitude

the temperature

bands.

to occur,

located

of the absorption

both

raising

occurs

than

exciton

the same

interpretation.

and wavelength,

band

in the wavelength

Since

are

the asymmetry

in exciton

arrangement

12 8 to 45 8.

extrema

at 574 nm rather

to be expected

coupling

spectrum

in amplitude

for

Moreover,

Interchromophore from

both

At the same

amounts.

A rather

required. range

in both

spectrum.

exciton

the first

support

and the two

occurs that

should

strongly

CD exciton

in (1) accounts

predicts

lobes

nm)

maximum

the crossover

in both

For

(567

superposition

the fact

RESEARCH COMMUNICATIONS

of a conservative

maximum respect

hand

AND BIOPHYSICAL

to be sa-

molecules the are

is

7 1 map

(6, 7),

proportional

to

the high

extinction

WAVELENGTH,nm

Fig. 2. The effect of solubilization in a 2 O/o (“/v) mixture of Triton X- 100 and 0.025 M phosphate buffer (pH 6.88) on the CD spectra of purple membranes from H. halobium. With increasing time after mixing with Triton X-100, the CD amplitudes decrease. Recording of the spectra was started at the following times after mixing: 2, 13, 30,46, 60, 80,125 and 200 minutes. Temperature 20° C.

899

Vol. 67, No. 3, 1975

coefficient

BIOCHEMICAL

at 567 nm

ces possible.

The

membrane,

since

Decoupling

(63 000 M-l

transition this

disassemble

the surface

If this

can be done

the exciton

coupling

accompanied

crease

minutes in light

amounts

molecules

and that

550 nm remained.

time

effect

X-100.

a period both

at the end of this This

is clearly

the spectrum.

Absorption

spectra

the CD spectra

showed

01 8 E z -2 zk

above

or loss with

hours.

time

may

take

by following

from

the de-

(Fig.

by approximately positive

in agreement

up to this

X- 100 is

of the absorption

We observed

a broad

were

band

and pH 6. 88 protein-micelle

decreased

which

in a detergent.

in Triton

solubilization

period

be to

the positive

on conditions,

peaks

for

of the chromophore,

and a shift

two

tests would

of the protein

and can be monitored

exciton

that

presented

temperature

distan-

of the

One of the simplest

Solubilization

of about

long

to the plane

in extinction

At room

over

to vanish.

to disappear

remaining.

hours,

scattering.

this

the exciton

expected

Depending

over

be parallel

denaturation

decrease

to severa.

occurs

during

are

coupling

cannot

by solubilization

serious

bands

to 550 nm.

formation that

lattice

RESEARCH COMMUNICATIONS

makes

of the CD spectrum

by a small

maximum

(12))

in Triton

without

due to the isolated

several

cause

by solubilization interpretation

cm-l

dipoles

would

the tentative

AND BIOPHYSICAL

with

our

measured

band

equal centered

interpretation simultaneously

no denaturation

2)

at of with

or chromophore

4 3

d 2 -f o‘; 1 E 0 * 5;1 -1 CL s -2 d

4 $

-3 400

500 WAVELENGTH,

600

700

nm

Fig. 3. The effect of temperature on the CD spectrum of a suspension of purple membranes from H. halobium in a 36. ‘7 O/o (“/v) mixture of dimethyl29.oOc; **** : sulfoxide and 0. 025 M phosphate buffer (pH 6. 88). -: 34.8’C; ---: 40. O°C; -. -. - : 45. O°C; : 50. ooc. Upon cooling from 50. O°C to 29. O°C the original spectrum at 29. O°C was obtained.

900

Vol. 67, No. 3, 1975

loss

BIOCHEMICAL

had occurred.

itself

Eventually,

by an extinction

absorption

band.

Reversible

changes

however,

decrease

(DMSO)/water

reversible

in the purple

bance,

shifting

DMSO

concentration,

velops

centered

produced

by altering

both

exciton

tive

band

positive

band

can be most mobility

the exciton

easily

explained

and translational

distances

between

Together

with

band

private

lattice

disappear

effects

at low

and at temperatures corded

of purple

At room

this

These

increases

state.

the addition

and a findings

the protein

Increased

rota-

to larger

average

of the CD amplitude.

could

easily

bacteriorhodopsin

the fact that

a conserva-

completely,

lead

and to the appearance

membrane

temperature

similar

which

temperatures.

ranging

to those

and the temperature, states,

DMSO

way,

to the com-

of a positive molecules.

the diffraction of DMSO

(R.

rings

This from

Henderson,

communication).

Bleaching

quite

upon

almost

fluid

mixture,

410 to 490 run.

to uncoupling,

shift,

bands

with

from

averaging

uncoupled

is in agreement

surface

lead

the simultaneous

at 460 nm due to isolated

the

would

and to angular

band

can be

in the CD

for

identified.

that

to a more

of the exciton

effect

de-

esta-

In a concerted

disappeared

proteins

disappearance

was

the change

develop

by assuming

mobility

plete

interpretation

have

leading

CD band

effect

as expected

at 460 nm can be clearly

in the membrane,

tional

amounts,

the

increasing

reversible

to 50°C.

a

in absor-

in a 36. 7 “10 DMSO/water

CD amplitudes bands

With

3 shows

29OC

by equal

changes

of this

the same

from

Positive

these

with

and a positive

reversibility

Fig.

of a 380 nm

spectrum,

with

disappear

suspensions

decrease

centered

absorption

concentrations,

is raised

structure.

At 55 O/o DMSO

peaks

manifesting

30 O/o and 60 O/o induce

in the CD spectra.

Complete

membrane

peaks

in,

by dimethylsulfoxide.

In parallel

the temperature.

the temperature

sets

between

membrane

changes

DMSO

of purple

when

major

at 460 nm.

At fixed

spectra

mixtures

the exciton

blished.

induced

to 460 nm (11).

we observed

denaturation

mobility

Dimethylsulfoxide

567 nm band

RESEARCH COMMUNICATIONS

at 550 nm and the appearance

in protein

change

AND BIOPHYSICAL

at room

from

Under

- 1OoC to - 30°C,

suspensions

the CD spectra observed

conditions

CD spectra

in 60 “10 glycerol/water in this

in water.

solvent Depending

the photo-equilibrium temperature

photostationary

between

lies

on the side

901

system

were

re-

mixtures. (Fig.

4) are

on the light

intensity

the 567 nm and 412 nm of the 567 nm state,

can

Vol. 67, No. 3, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

WAVELENGTH.nm

Fig. 4. The effect of bleaching on the CD spectra of a suspension of purple membranes from H. halobium in a 60 O/o (“/ v ) mixture of glycerol and 0. 025 M phosphate buffer (pH 6. 88). The spectrum at - 30°C was recorded under photostationary conditions with most of the bacteriorhodopsin molecules bleached. The spectrum at + 25OC refers to the unbleached light adapted state.

be shifted lived

towards

the 412 nm state

intermediates

cussion

to consider

amplitude optical

exist

(13),

only

these

two

bands

near

of the exciton activity

develops

the appearance bands

vations

can be explained

bands

band

bleached

lead

for

isolated

unbleached

even

interpretation, dence

are

coordinated

at 412 nm, positive

band

At low some

light

CD bands,

since

having

light shown

There

disappearance

will levels in Fig.

experiments

a single

neighbor

effects

lead

to the broad

band

not all molecules

are

4 appear

exciton

902

The sign

hand,

however, peaks,

CD

The

no

the probabihigh

enough

remaining

at 567 nm,

since

bleached.

Although

to be in agreement

on the intensity

obser-

on the average

becomes

at 412 nm.

to

the exciton

have

a bleached

leads

These

bleached.

have

time

at 567 nm the

are

on the other

can be no doubt of both

they

dis-

the

further

4).

intensities

of exciton

molecules

further

light

level

intensities,

to exciton At high

of this

and at the same

(Fig.

molecules

shorter

intensities,

whereas

can only

data

needed.

the light

at 412 nm,

and high

the experimental

light

absorbing

the observation

at - 30°C

band

since

molecule

to allow

At low

Raising

other

the purposes

567 nm is reduced

as follows.

neighbors.

of a bleached

several

for

states.

by a weak

reduced,

molecules,

and cannot

type

replaced

at 567 nm are

bleached

lity

are

Although

it is sufficient

at 412 nm.

of an exciton

exciton

(12).

with

and temperature that bleaching

this depen-

leads

and to the appearance

to a of

Vol. 67, No. 3, 1975

optical has been

activity well

BIOCHEMICAL

in the bleached exposed

state.

in the bleaching

AND BIOPHYSICAL

The

underlying

process,

RESEARCH COMMUNICATIONS

positive

supporting

band our

at 567 nm

interpretation

of the CD spectrum.

REFERENCES 1. Abrahamson, E. W., and Fager, R. S. (1973) Current Topics in Bioenergetics, 5, 125-200. 2. Poo, M., and Cone, R. A. (1974) Nature, 247, 438-441. 3. Blaurock, A. E., and Stoeckenius, W. (1971) Nature New Biol., 233, 152155. 4. Henderson, R. (1975) J. Mol. Biol. I 93, 123-138. 5. Blaurock, A.E. (1975) J. Mol. Biol., 93, 139-158. 6. Unwin, P. N. T., and Henderson, R. (1975) J. Mol. Biol., 94, 425-440. 7. Henderson, R., and Unwin, P. N. T. (1975) Nature, 257, 28-32. 8. Becher, B. M., and Cassim, J. Y. (1975) Abstr. 19th Ann. Meet. Biophys. Sot. W-PM-F3. 9. Oesterhelt, D., and Stoeckenius, W. (1971) Nature New Biol., 233, 149152. 10. Heyn, M. P., J. Phys. Chem., in press. 11. Oesterhelt, D. , Meentzen, M. , and Schuhmann, L. (1973) Eur. J. Biothem., 40, 453-463. 12. Oesterhelt, D., and Hess, B. (1973) Eur. J. Biochem., 37, 316-326. M. (1975) Biophys. Struct. Mech., 1, 259-271. 13. Dencher, N., and Wilms,

903