Light dependent and biochemical properti

() Photochemistry and Photobiology, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA1999, 69(5) 599 604 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Light dependent and Biochemical Propert[.]

Photochemistry and Photobiology, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 1999, 69(5): 599-604 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

Light-dependent and Biochemical Properties of Two Different Bands of

Francesco Lopezi, Simona Lobasso2, Matilde Colella2, Angela Agostiano113 and Angela C ~ r c e l l i * * , ~

'Dipartimento di Chimica and zDipartimento di Fisiologia Generale ed Ambientale, Universita di Bari, Bari, Italy and

3Centro Studi Chimico Fisici sull'lnterazione Luce-Materia, CNR, Bari, Italy

Received 4 January 1999; accepted 19 February 1999

We report a detailed description of the light-dependent

and biochemical properties of two different bands of iso-

lated and nearly delipidated bacteriorhodopsin obtained

from chromatography on phenyl-Sepharose CL4B The

two bands (BR I and BR 11) showed a number of mark-

edly different spectroscopic and biochemical character-

istics: different absorption maximums in the dark, dif-

ferent lighvdark adaptations, different M decay kinetics,

different stabilities, different responses to titration with

alkali in the dark and different circular dichroism (CD)

spectra Organic phosphate contents of BR I and BR I1

were measured; we found that more than 90% of purple

membrane organic phosphate was removed in the course

of chromatography and that the phospholipidprotein

molar ratio was always higher in BR I than in BR 11 In

many functional aspects (high stability, response to light

adaptation, spectral changes in the dark by alkali addi-

tion and bilobate CD spectrum) the first band appeared

to be similar to the purple membrane We suggest that

the functional differences between the two bands depend

on the fact that the first band (BR I) contains mostly

bacteriorhodopsin aggregates corresponding to purple

membrane trimers, while the second band (BR 11) con-

tains only bacteriorhodopsin monomers

Solubilization and isolation of lipid-depleted bacteriorhodop-

sin (BR)? was first achieved by Wildenauer and Khorana

(1) Solubilization of the purple membrane (PM) with Triton

X-100 followed by gel filtration on agarose in deoxycholate

solution resulted in removal of 99% of endogenous lipids zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

( 2 ) Solubilization with octylglucoside followed by agarose

*To whom correspondence should be addressed at: Dipartimento di

Fisiologia Generale ed Ambientale, UniversitA degli Studi di Bari,

Via Amendola 165/a 70126 Bari, Italy Fax: 39 80 544 3388;

e-mail: a.corcelli@biologia.uniba.it

tdbbreviations: BR, bacteriorhodopsin, BR I, first eluted band from

phenyl-Sepharose chromatography; BR 11, second eluted band

from phenyl-Sepharose chromatography; CD, circular dichroism;

DA, dark-adapted; LA, light-adapted; OG, n-octyl-P-glucopyra-

noside; PGP-Me, phosphatidylglycerol phosphate methyl ester;

PGS, phosphatidylglycerol sulfate; PM, purple membrane zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

chromatography has been used to obtain delipidated BR used

in recent crystallization studies (3,4) Another method zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA of BR isolation has been developed using high performance size

exclusion chromatography (5) A partial characterization of delipidated BR properties in various detergents has been also

obtained by Milder et al (6) Here we report data illustrating

the characteristics of bacteriorhodopsin isolated by means of

phenyl-Sepharose C L 4 B chromatography As previously re-

ported for halorhodopsin zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA(7,8), we show that BR also splits

into two bands on phenyl-Sepharose and that these bands have a number of remarkably different light-dependent and biochemical properties The appearance of two BR bands on phenyl-Sepharose C L 4 B was previously mentioned (8) Re- cently, other investigators who solubilize wild-type and mu- tant BR for the purpose of crystallizing it have observed that two bacteriorhodopsin peaks come off from the phenyl-Se- pharose column (J K Lanyi, personal communication), but

in the literature there are not other studies illustrating the different properties of the two bands or explaining what the splitting represents Although the behavior of BR in micellar systems has been described in the past (9,10), in this paper

we present new data illustrating in detail the properties of solubilized BR in octylglucoside and, furthermore, we show that with this new isolation method it is possible to isolate and distinguish nearly delipidated BR fractions in the form

of monomers and trimers

MATERIALS AND METHODS

Materials An engineered L33 Halobacterium salinarum strain, kindly provided by Richard Needleman, was used in this study (1 1)

The growth medium, containing neutralized peptone (L34, Oxoid)

and novobiocin (from Serva, at final 1 pg/mL), was prepared as

previously described ( 12) DNase and n-octyl-P-glucopyranoside (octyl glucoside or OG) were obtained from Sigma, sodium cholate from Serva and phenyl-Sepharose CL-4B from Pharmacia

PM isolation Purple membrane was isolated as previously de-

scribed (13) A concentrated suspension of cells in 4 M NaCl was

dialyzed overnight; the dialysate was washed four times with dis- tilled water by centrifuging and the pellet (in distilled water) was layered over a step gradient (60% 3 5 8 , and 15% sucrose) and cen- trifuged 18 h 100000 g The purple band was collected and sucrose was removed by repeated washing Finally, the collected PM was frozen (-20°C)

BR isolation Membrane solubilization was performed by incu-

bating 12 mg of PM at room temperature for 64 h with zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA5 mL 0.1

M Na acetate, 1% Triton X-100 (pH 5 ) An aliquot of the extract (about 55 nmol of BR) was diluted 10-fold with 0.4% sodium cho-

late buffer containing 0.1 M NaC1 buffered with 25 mM Tris/HCl (pH 7.2) and then loaded (0.2 mL/min) over a 1 X 3 cm column of

599

600 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Francesco Lopez zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA et a/

0.4

n

C

E zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

0 0.3 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

0

0 5 fractions 10 15 20 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

Figure 1 Chromatography of BR on phenyl-Sepharose CL-4B gel

BR was solubilized in 1% Triton X-100 diluted in cholate buffer to

obtain absorption on phenyl-Sepharose during loading and eluted in

0.5% octyl glucoside buffer (pH 7.2)

phenyl-Sepharose CL-4B (2.5 mL bed volume) previously equili-

brated with the same buffer I t was washed with 70 mL of cholate

buffer (0.8 mL/min) and then eluted with octyl glucoside buffer

containing zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA3 M NaCI 25 mM Tris/HCI (pH 7.2) and zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA0.5% octyl

glucoside (0.3 mL/min)

Chromatography was performed at room temperature under dim

light; collection of the colored fractions ( I -5 mL) started after elution

of about 12 mL of octyl glucoside buffer Total recovery of BR

from the column was 90-100%% The absorptions at 560 and 280 nm

of each BR-containing fraction were measured spectrophotometri-

cally (A28dA560 1.3-1.6): BR fractions were stored in the dark at

-20°C The same chromatography was repeated at pH 5 using 0.1

M sodium acetate buffer instead of Tris/HCl in the washing and

eluting solutions

Pho.~phorus qicrintitatictn The content of organic phosphorus was

measured using the method of Bartlett (14)

Ab.rorptinn spectrosc-opy UV-visible absorption spectra were ob-

tained with a Cary 3 UV-visible spectrophotorneter Dark-adapted

( D A ) spectra were obtained on samples stored at room temperature

in complete darkness for 12 h Light adaptation of PM and BR was

accomplished using a 150 W illuminator A cuvette containing sam-

ple was irradiated for 5 min at a distance of 15 cm from the bulb

The rate of dark adaptation of BR was measured by following ab-

sorption decrease at the wavelength in the visible region with the

largest absorption difference between light-adapted (LA) and DA

forms

M decay The kinetics of M decay was followed using an Optical

Multichannel ‘4nalyzer (OMA 111, model 1460 EG&G Princeton,

N Y ) equipped with an EG&G Red Intensified Diode Array Detector

model 1430-512-G Actinic flashes were provided by an EG&G xe-

non lamp (3.35 J discharge energy) and screened through two layers

of Wratten gelatin filter giving a light pulse of 4 ps duration at half-

maximal intensity The probe beam was screened with a blue (300-

500 nm) filter Deconvolution of multiexponential decays was per-

formed by computer routines based on the Marquardt algorithm

Samples for the kinetic studies were thermostatted (20°C) All sam-

ples were LA before measurements were taken

Circulur dichroisnt Circular dichroism (CD) spectra were ob-

tained with a Jasco spectropolarimeter; BR samples in octyl gluco-

side buffer having absorption visible in the range 0.5-0.8 were an-

alyzed

RESULTS

BR splits in two bands in phenyl-Sepharose CL4B

chromatography

We isolated two different bacteriorhodopsin bands on phe-

nyl-Sepharose gel by using essentially the same protocol de-

veloped years ago by Duschl et al ( 15) to isolate and purify

halorhodopsin Purple membranes were solubilized in Triton

and diluted with cholate buffer to obtain BR binding on phe-

nyl-Sepharose BR was extensively washed on gel with cho-

0.12

Q)

* 0.1

3

s: 0.06 0.08

P

0.04 0.02

0

4

b :

350 450 550 650

AA 0.02 0.01

0 -0.01

350 450 550 650

nm

Figure 2 Dark (solid line) and light (dotted line) adapted spectra

of (a) BR I and (b) BR 11 c: Light-dark difference spectra of BR I (dotted line) and BR I1 (soliddotted line) are compared with the difference spectrum of PM in water (solid line)

late buffer to remove PM lipids and then eluted by shifting

to octyl glucoside buffer

Figure 1 illustrates the chromatographic profile of solu-

bilized BR eluted on phenyl-Sepharose CL-4B in octyl glu- coside buffer at pH 7.2 It can be seen that two different BR bands are obtained (BR I and BR 11) and that BR I represents about 30% of total loaded protein

The ratio between the areas of the two bands does not change when half or double protein amounts are loaded on the column Furthermore, the profile was not significantly modified by running the chromatography at pH 5 At lower

pH the protein tends to remain bound to the gel longer and very broad bands are obtained In the following we illustrate the biochemical and spectroscopic characteristics of the two bands obtained at pH 7.2 No difference in the chromato- graphic profile was found between isolation procedures per- formed in room light and in the dark

Light/dark adaptation and M decay of the two bands

Figure 2a,b illustrates spectra in the dark and after light ad- aptation of BR I and BR I1 isolated at pH 7.2 at 22°C Re-

markable differences in the dark spectra as well as in the light adaptation phenomena of BR I and BR I1 can be seen

In the dark, the absorption maximum of BR I is at 555 nm, while the absorption maximum of BR I1 is blue-shifted at about 552 nm Furthermore, in the case of BR 11, besides the 552 nm peak another peak at 380 nm is present; the

Photochemistry and Photobiology, 1999, 69(5) 601 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

Table 1 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

lized PM and BR

Lightldark absorbance maximum of PM, Ttiton-solubi-

(nm) (nm) (nm) %

PM 5 60 568 8 f l l

Solubilized PM 550 558 8 -6

BR I 555 562 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAI zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAt9

“da = dark-adapted

tla = light-adapted

height of this peak increases together with a decrease of

absorption in the visible region when BR 11 is left at room

temperature even for only a few hours The spontaneous

conversion of BR into BR I1380 can be reversed by low-

ering the pH to 5 (not shown) BR I is instead quite stable

and has only a negligible tendency to form the 380 nm spe-

cies By keeping the sample at 0°C in the dark, either no

formation of the 380 nm species or no increase of the 2801

560 absorption ratio was observed for BR I over a period of

3 months from isolation, while in the same time the second

band showed a 30% decrease in absorption at the peak in

the visible region

Interestingly, the extent of light adaptation of BR I is sim-

ilar to that of PM, as after illumination a red shift of ab-

sorption maximum is observed, together with an increase in

the absorption value (of about 10%); in contrast, light ad-

aptation of BR I1 is considerably reduced The similarity in

light adaptation between BR I and PM is also evident from

an examination of difference spectra of BR I and BR I1

reported together with the difference spectrum of PM in wa-

ter in Fig 2c On the other hand, the light-dark difference

spectrum of BR I1 resembles that of alkalinized PM (not

shown in Fig 2C)

Table 1 reports dark and light lambda max and the per-

centage changes in absorptions after illumination of PM, Tri-

ton solubilized PM and the two BR bands; the values are

means obtained from several different spectra The return to

the dark state is also different for BR I and BR 11 The

process of dark adaptation for BR I can be well described

by a biexponential equation, while dark adaptation of BR I1

is slower and difficult to fit, probably because it occurs to-

gether with a loss of absorption, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAi.e with some protein deg-

radation due to instability of this BR fraction (see Fig 3)

Figure 4 illustrates the M decay of both BR I and BR 11;

it can be seen that M decay of both BR forms is slower than

that of PM Since BR I and BR I1 are expected to be deli-

pidated as a consequence of the isolation procedure (see be-

low), this result is in agreement with previous data obtained

for delipidated PM (16,17); in both cases the traces are well

fitted by biexponential equations whose life times are given

in Fig 4 Furthermore, it can be seen that M decay of BR

I1 is faster than that of BR I Literature data describing the

dependence of M decay kinetics on the flash intensity and

the cooperative effect caused by a different excitation of

monomers and trimers in BR can help to interpret this result

(18), but our data are too preliminary to allow conclusions

on the differences in the photocycle intermediates of BR I

and BR 11 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

r

3

0.8

0.6 0.4

0.2

h

e

0 50 100 150 200

time (min)

Figure 3 Kinetics of dark adaptation of ( 0 ) BR 1 and zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA( 0 ) BR 11

Both samples were at concentrations of about 3.5 k M

Different responses to alkali addition in the dark

Figure 5 (a,b) shows the absorption spectra of BR 1 and BR

I1 as a function of pH in the pH range 7.2-11; the pH was

adjusted in the dark by addition of 1-5 pL aliquots of 0.1 N

NaOH Figure 5 (c-f) shows the difference spectra between the high pH spectra and the spectrum at pH 7.2 During the titration, the formation of three peaks at 460, 380 and 365

nm can be seen for BR I, while only the 380 and 365 nm peaks are seen for BR 11

In a narrow pH range, the spectral changes induced by alkalinization can be partially reversed for both BR I and

BR I1 upon acidification (not shown) Furthermore, by plot- ting residual absorption in the visible region at increasing

pH, it can be seen that BR I is more resistant to alkali ad- dition than BR II; in fact, bleaching of BR I is complete at

pH 11, while BR I1 is already completely bleached at pH 10

(see Fig 6)

As expected, the accessibility of Schiff base to external

OH- is greatly increased in both BR I and BR I1 compared

with PM Furthermore, these results indicate that BR I also shows characteristics similar to those of PM in the titration

with alkali, while the inability of BR I1 to give rise to the

460 nm species upon alkalinization offers another example

of similarity of this band to the so-called “alkaline BR”

(19,20) The functional similarity between alkaline BR and

BR I1 prepared by delipidation of BR could be due to the

fact that PM titration with alkali could induce a weakening

of the lipid-protein interactions mimicking the delipidation effects in BR 11

n

c

E

1

0.8

0.6 0.4 0.2

0

0 100 200 300 400 500

time (ms)

Figure 4 Comparison of the M decay kinetics of BR I (solid line)

and BR I1 (dotted line) after flash excitation The samples were at the following concentrations: BR I, 4.3 pM BR 11, 7 KM

602 0.1 Francesco Lopez zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAet zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAa/ zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

Y

2 0.0s zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

2 0.06

2 0.04

2 0.02

a, 0.02

0.01

0

a -0.02 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

3 -0.03

-0.04 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

8 0.08

0.04

f -0.01

f0, o

2 -0.04

-0.08

400 500 600 400 500 600

Figure 5 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAa and b: Abwrption spectra of ( a ) BR I and t b ) BR I1

samples as a function of pH at room temperature The samples were

DA at pH 7.2 and then adjusted to a given pH by the addition of

NaOH I n the order o f decrease in absorption at the maximum: a:

I pH 7.1: zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA1 pH 7.58: 3 pH 8.02; 4 pH 8.39: zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA5 , pH 8.87; 6, pH

9.27: 7, pH 10.29: 8 pH 10.67: 9 pH 10.81: 10 pH 11.04, b: 1,

pH 7.1; 2 pH 7.61 3 pH 8.07: 4, p H 8.41: 5 pH 8.92; 6 pH 9.31;

7, pH 10.29: 8 , pH 10.68 c-f: Difference absorption spectra of ( c

and ci BR I and (ct and f ) UR I1 samples at pH, minus that at pH

7.2 c : I, pH, 7.58: 2 pH, X.02; 3 pH, 8.39; 4 pH, 8.87 d: 1 pH,

7.61: 2 pH, 8.07: 3 pH, 8.12: 4 pH, 8.91 e: 5 pH, 9.27; 6, pH,

10.39: 7 pH, 10.67: 8 pH, 10.87: 9 pH, 11.04 f 5 pH, 9.32; 6

pH, 10.19: 7, pH, 10.68 Optical densities of both BR I and BR I1

at 550 nni in the dark ;it p H 7.2 were 0.09: pathlength of the cuvette

I cm

Residual phospholipids associated with the two bands

Another important question to address is that of the lipid

content of BR fractions eluted from the phenyl-Sepharose

column Table 2 reports the lipid phosphorus content togeth-

er with the estimated phospholipid/BR molar ratio for the

two bands The ratio values have been obtained taking into

consideration that about 80% of PM phospholipids contain

two phosphate groups per lipid molecule (as in the case of

phosphatidylglycerol phosphate methyl ester [PGP-Me]) and

7 8 9 10 11

PH

Figure 6 Titration curves of (*I BR I and ( 0 ) BR 11 Relative

absorption i n the visible region is plotted \'ersii.s increasing pH: data

of t w o diffcrcnt titraiion experiments are reported

Table 2

PM and BR*

Organic phosphorus content of PM, Triton-solubilized

Phosphorus BR PhospholipidslBR (nmol) (nmol) molar ratio

PM 150.3 -C 6.0 16 5.7 Solubilized PM 197.9 C 0.0 18 6.4

BR I 31.9 2 8.5 18 1.1

BR I1 21.4 2 7.5 29 0.5

( 1 2 )

( 4 )

( 6 )

( 6 )

*Phosphorus content is given as mean C SD of different determi- nations (number reported in parentheses) BR amount has been

estimated by using A,,, ( e = 6 3 000) for PM, A560 (E = 54 000) for Triton-solubilized PM and A,,,, ( e = 66000) for BR 1 and

BR 11 The phospholipidsBR molar ratio has been estimated con- sidering that 30% of total phospholipids is represented by phos- phatidylglycerosulfate (1 P/lipid) and the remaining 80% by

phosphatidylglycerolinethylphosphate (3 P/lipid)

that the remaining 20% contains only one phosphate group per lipid molecule (as in the case of phosphatidylglycerol sulfate [PGS]) At least one phospholipid molecule is still

associated with BR I, while only one phospholipid per two

BR molecules are available on average in BR 11

CD

As BR I was found to have light adaptation similar to that

of PM, we thought it reasonable to check for the presence

of an exciton coupling effect in the CD spectrum of BR I

Figure 7a,b shows the CD spectra of BR I and BR 11 We readjusted the isolation protocol in ordcr to obtain more con- centrated BR I samples suitable for CD analyses in the vis- ible region Both samples used in the experiment in Fig 7

had absorbance at 560 nm of about 0.7; attempts to obtain

more concentrated BR I fractions were unsuccessful Despite

the noise, it can be seen that the CD spectrum of BR I is

clearly bilobate, indicating interaction between retinal chro- mophores on adjacent BR molecules, as it likely occurs in

PM trimers However, the possibility that the exciton cou- pling of BR I could be due to aggregates different from trimers (in particular, dimers) cannot be excluded (21)

Only a positive band is instead present in the CD spectrum

1.5

-1.5

450 500 550 600 650 450 500 550 600 650

Figure zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA7 Visible CD spectra for (a) BR 1 and (b) BR 11 Optical

densities at 560 nm of BR I and BR I1 were, respectively 0.685 and 0.616

Photochemistry and Photobiology, 1999, 69(5) 603 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

Table 3 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

of BR I and BR I1

Summary of the biochemical and spectroscopic properties

Phospho-

CD l i p i d s / Dark Light bilobate Stability BR

BR I zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAh red shift Yes High 2 1

BR I1 A,,, 555 nm red shift AA > 0 No Low < I zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

blue-shifted AA < 0

of BR I1 (Fig 7b); it can be noted that the positive BR I1

CD band is centered at 525 nm and not at the absorption

maximum of BR zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA I1 (552 nm dark, 560 nm light; see Table

1) We believe that this spectrum represents the sum of a

broad positive band of the monomers with a contribution of

the exciton band of trimers as a consequence of some over-

lapping of the two bands eluted by the column The rela-

tively low ellipticity of BR I could be an intrinsic charac-

teristic of the delipidated aggregates present in this fraction

as a result of the detergent exposure and the loss of the

native lipid environment zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

DISCUSSION

We developed a procedure to isolate two different fractions

of BR by means of phenyl-Sepharose CL-4B chromatogra-

phy Table 3 summarizes the functional and biochemical dif-

ferences between BR I and BR 11 BR I and BR I1 showed

different absorption spectra in the dark, different lightldark

adaptations, different M decay kinetics, different stabilities,

different responses to titration with alkali in the dark and

different CD spectra No difference in the protein moiety

sizes of BR I and BR I1 was found by means of sodium

dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-

PAGE) analysis (not shown); therefore, we can rule out that

the functional differences present in BR I and BR I1 are

consequences of the presence of different processed BR mol-

ecules in the purple membrane

The high stability, the response to light adaptation, the

titration experiments and the presence of an exciton coupling

band in the CD spectrum of BR I illustrate the similarity of

BR I to BR in the PM and therefore strongly suggest the

presence of BR trimers in BR I At the same time most of

the functional data reported here are consistent with the pres-

ence of monomers in BR 11

After delipidation, on average, one very tightly bound

phospholipid remains associated with BR I The amount of

residual phospholipids associated with BR I is compatible

with the aggregation of three BR molecules and three phos-

pholipid molecules into trimers, presumably with one phos-

pholipid molecule in the crevice between monomers, which

also occurs in PM, as suggested by electron (22) and neutron ( 2 3 ) diffraction data This result strongly suggests a role for zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

PGP-Me andlor PGS in trimer formation It is interesting

here to note that a specific requirement for either PGP-Me

or PGS in the two-dimensional hexagonal array formation

of BR was previously demonstrated in reconstituted systems

(24)

However, based on the data of the present study, we can-

not exclude the presence of residual glycolipids in the both

BR I and BR 11 It has been reported that only 45% of gly- colipids are removed by treatment of PM with Triton X-100

(25) Furthermore, recent data have indicated that both phos- pholipids and glycolipids are associated to delipidated BR

fractions isolated with a different isolation method and used

in crystallization studies (26) BR I and BR I1 could repre- sent two different BWphospholipid complexes or aggregates already present in different proportions in the Triton solu- bilized membranes as a result of incomplete solubilization

or as a consequence of an intrinsic heterogeneity of the PM

This last possibility would be suggested by the fact that the ratio between the areas of BR I and BR I1 was found to be constant by changing the experimental conditions, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBAe.g by lowering the pH of chromatography buffers, by increasing

the Tritodprotein ratio in the solubilization step or by chang- ing the conditions of dilution with cholate (not shown)

However, it is also possible that although a basic homoge- neity is present in the PM, the two different species of BR

are produced in the course of the delipidation process due

to the fact that it is extremely difficult to remove the last remaining phospholipid and that what is recovered is the residual last two components remaining in the conversion of

BR I to BR I1 by phospholipid removal The nature of the residual phospholipid(s) bound to BR I and BR I1 is pres- ently under investigation

In conclusion, the BR isolation method described in this report offers the advantage of separating monomers and tri- mers of BR, resulting in final homogeneous BR preparations, which can be potentially useful in crystallographic studies

Ackizon,ledjieineizt~~-We are grateful to Alberto Spisni and Cesirn

De Chiara of the Centro Interdipartimentale Misure of the University

of Parma for CD measurements and Emanuele Carulli for technical assistance This work was supported by Italian Minister0 dell' Univ- ersita e della Ricerca Scientifica e Tecnologia (MURST) and Con- siglio Nazionale delle Ricerche (CNR)

REFERENCES

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