Journal of Photochemistry and Photobiology B: Biology 58 (2000) 163–169
The effect of lipid environment in purple membrane on bacteriorhodopsin
Kunsheng Hu , Ying Sun, Deliang Chen, Yinan Zhang
Institute of Biophysics Academia Sinica, Beijing 100101, China
Received 11 May 2000; accepted 9 October 2000
The decay rate of the Bacteriorhodopsin (BR) photocycle intermediate M
ratios of M to other intermediates and the rotational correlation time (t ) in purple membrane (PM) fragments treated by the
zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) with different concentrations were
studied. The results show that: (1) The largest effect of CHAPS on M
of Mto other intermediates in BR photocycle is in the range of its critical micelle concentration (CMC). This indicates that changes of
the ratios of Mto other intermediates, t , M decay and proton decay occur and are due to the variation of the lipid environment. (2)
The dependency of proton yield on CHAPS concentrations is basically consistent with that of M
proton pumping function and M. These studies show the importance of maintaining a native environment.
B.V. All rights reserved.
and proton, the proton pump efficiency (H /M), the
decay rate and proton decay rate of BR, t of PM and the ratios
. This indicates the relation between
2000 Elsevier Science
Keywords: Bacteriorhodopsin; Purple membrane; CHAPS
has several values measured by different laboratories [7,8].
There are many factors that affect kinetics of M
and protonation, such as lipid in PM, cations, temperature,
pH, etc. One of the good methods to study the mechanism
of BR photocycle is to use detergent to remove lipid in PM
[9,10]. Different detergents have different effects on BR
and lipid in PM. Nonionic detergent Triton X-100, b-
Octylglucoside, bile salt [11–13] can remove lipid in PM
to different extents and change the kinetic process of BR.
About the effect of zwitterionic detergent CHAPS on the
structure and function of PM and BR, few papers can be
The effect of different environment on the secondary
structure shows detergent solubilization results in changing
the protein conformation . There are relationship
between structure and function on BR, the change of
structure should affect the BR photocycle process and
protons pump function. This paper presents the effect of
zwitterionic detergent CHAPS on the rotational correlation
time (t ) in PM, the intermediate M
BR photocycle, the proton decay process, the ratios of
Mto other intermediates and the proton pumping
function resulting from changing lipid environment in PM.
From these results it shows there is a relation between
Bacteriorhodopsin (BR), the only protein in Purple
Membrane (PM), has the function of a light-driven elec-
trogenic proton pump. The protein consists of 248 mainly
hydrophobic amino acids and a chromophore, all-trans-
retinal, bound to lysine-216 via a protonated Schiff-base
linkage [1,2]. Upon the absorption of yellow light, BR
goes through a photochemical cycle consisting of several
distinct spectroscopic intermediates, K
[3,4]. Among them, the formation and decay of M
associated with proton pumping function . In the
process of M formation, M
rise components . Mdecay was also confirmed to
have two components, a fast decaying component of M
(M ) and a slow decaying component of M
The event linked with the BR photocycle is the proton
pumping function. The quantum yield of the photocycle
, L, M, O
was observed to have two
decay process in the
*Corresponding author. Tel.: 186-10-6488-8580; fax: 186-10-6487-
E-mail address: email@example.com (K. Hu).
1011-1344/00/$ – see front matter
2000 Elsevier Science B.V. All rights reserved.
K. Hu et al. / Journal of Photochemistry and Photobiology B: Biology 58 (2000) 163–169
proton pumping function of BR and M
largest effect of CHAPS on BR photocycle is in the range
of its critical micelle concentration. Lipid environment is
important for the structure and function of purple mem-
3.1. The steady-state absorption of BR treated with
The absorption maximum of BR treated with different
concentrations of CHAPS is blue shifted by 2–7 nm (Fig.
1). The absorption in the UV range has little change. This
shows that the retinal chromophore is affected by adding
2. Materials and methods
CHAPS was purchased from Sigma. For growth of H.
halobium and isolation of purple membrane, the method of
Oesterhelt  was used with slight improvement. The
purple membrane fragments were suspended in various
CHAPS concentrations (0–40 mM). The mixed membrane
suspension was adjusted to pH56.7 and OD (568)50.2,
then incubated for 24 h at 48C in the dark. For the ESR
measurements, purple membrane was labeled with 16-
doxyl-stearic acid, free radical (from Sigma) for 3 h at
room temperature, then the labeled sample was washed
several times until the free spin label was removed. ESR
spectra were obtained from Varian E-109, X band, center
magnetic field, 3200 Gs, sweep width 200 Gs, time
constant 0.1280 ms, sweep time 120 s, microwave power
20 mW, modulation amplitude 1 Gs, microwave frequency
9.13 GHz. The steady-state absorption spectra was ob-
tained from an UVIKON-930 UV/visible spectrometer.
The ratios of M to other intermediates in the photocycle
were determined by laser Raman spectroscopy (JY T-800).
The excitation wavelength was 488 nm. BR treated with
CHAPS was illuminated in front of a 250 W tungsten lamp
(10 cm) for more than 1 min before the start of the
experiment for Raman measurements, flash kinetic spec-
troscopy measurements and the steady-state absorption
measurements. All measurements were done at 2560.18C
in a circulating water bath with temperature control (RTE-
8, NESLAB). The kinetics of M
at the wavelength 412 nm and proton decay were assayed
by probing the transient absorbance changes of illuminated
BR using an average of 85 flashes at 400 nm. Transmission
changes of BR and pH indicating dye were measured with
a single beam flash kinetic spectrophotometer constructed
in our laboratory. The measuring light source was a 250 W
tungsten lamp. Illumination was induced by a photographic
flash-lamp (BY-18) made by a camera manufacturer in
Shanghai with a 0.4-ms flash duration. The flash beam was
passed through a yellow filter (OG4). The wavelength of
412 nm will be cut off, while the wavelength of 568 can
pass through. The change in proton concentration of the
assay medium (H /mol BR) was calibrated in 0.1 mM
HCl with p-nitrophenol as a dye indicator using continuous
illumination. The differential extinction coefficient is as-
sumed to be 23 mMcm
Proton yield is calculated from the ratio of protons released
per M (H /M).
3.2. Kinetics of Mand proton decay
Changes were measured in the decay kinetics of inter-
mediate Mand proton in BR treated with CHAPS. For
BR treated with 6 mM CHAPS concentration (this con-
centration is in the range of CHAPS critical micelle
concentration), the decay times of M
crease obviously (Fig. 2). The half times of M
and proton decay (t
) as functions of CHAPS con-
centration are shown in Fig. 3. The t
increase from 3.8 to 43 ms and from 6 to 97 ms,
respectively, when CHAPS concentrations increase from 0
to 20 mM. The decay half times of proton and M412s
exhibit a sharp increase from about 2 mM CHAPS. At high
CHAPS concentrations, the increase of both t
saturate. This phenomenon indicates that the effects of
zwitterionic detergent CHAPS on the decay of M
proton are strongly related with its effects on the lipid
environment of BR .
and proton in-
3.3. Proton pumping function
Between 2–4 mM CHAPS, the proton yield (H /M
of BR in 0.1 M KCl was the lowest, 0.67, which was much
less than natural BR (Fig. 4). H /M
above 4 mM CHAPS, but it was still less than that of
natural BR. It is suggested that the proton pumping
began to increase
decay rate was detected
at wavelength: 412 nm .
Fig. 1. CHAPS concentrations dependence of absorption maximums of
BR treated with CHAPS at pH56.7, temperature: 2560.18C.
K. Hu et al. / Journal of Photochemistry and Photobiology B: Biology 58 (2000) 163–169165
Fig. 4. CHAPS concentrations dependence of proton yield H /M
and M(n) in 0.1 M KCl at pH56.7 and temperature: 2560.18C.
intermediates . I
tive quantity of M
Table 1. When the concentration of CHAPS was near 6
mmol, the ratio of I /I
that near the critical micelle concentration of CHAPS, the
relative quantity of Min photocycle reached the maxi-
is due to the C=C stretching vibration of other
in the photocycle. The relationship of
cmand CHAPS concentrations was shown in
could show the rela-
cm was largest. It means
Fig. 2. Comparison of M
For native BR. (b) For BR treated with 6 mM: CHAPS. Temperature:
2560.18C; Probe wavelength: 400 nm for proton decay and 412 nm for
decay of BR with those of proton decay. (a)
3.5. ESR spectra
function is also dependent on the lipid environment in PM.
Moreover, the dependency of proton yield on CHAPS
concentrations is basically consistent with that of M
on CHAPS concentrations (Fig. 4). This indicates the
relation between proton pumping function and M
Fig. 6 shows the ESR spectra of PM treated with 0–40
mM CHAPS labeled by 16-doxyl-stearic acid, free radical.
It is shown that in the ESR spectra, the parameters which
are used to calculate the rotational correlation time have
changed when PM is treated with successive concentration
of CHAPS. Fig. 7 shows the effect of CHAPS on
rotational correlation time t of hydrophobic inner area in
PM. The t increased with CHAPS concentration increas-
ing, when CHAPS concentration was 5 mM, t reached the
3.4. Raman spectra
Fig. 5 shows the Raman spectra of BR treated with 0–16
mM CHAPS. The band at 1567 cm
stretching vibration of M intermediate, the band at 1530
is due to the C=C
The data above demonstrates that CHAPS has effects on
the kinetics of Mdecay and proton pumping function.
The zwitterionic detergent CHAPS is known to be effec-
tive at solubilizing lipids and membrane proteins without
denaturation of proteins . Since the CMC of CHAPS
[14,20,21], the range of CMC is from 4 to 10 mM CHAPS
concentration. In this experiment, the effect of CHAPS on
purple membrane is to solubilize the lipids around BR first,
for lipids are solubilized more easily than proteins. CMC
of the detergent provides not only a guide for the appro-
priate concentration of detergent, but also an approximate
upper limit to the concentration of membrane to be
Fig. 3. CHAPS concentrations dependence of t
in 0.1 M KCl at pH56.7 and temperature: 2560.18C.
(1), and t
K. Hu et al. / Journal of Photochemistry and Photobiology B: Biology 58 (2000) 163–169
Fig. 5. Raman spectra of BR treated with 0–16 mmol/l CHAPS. (a) 0 mmol/l; (b) 2 mmol/l; (c) 4 mmol/l; (d) 6 mmol/l; (e) 8 mmol/l; (f) 10 mmol/l;
(g) 12 mmol/l; (h) 14 mmol/l; (i) 16 mmol/l.
solubilized. The process of liposome solubilization and
reconstitution of bacteriorhodopsin have been studied
using CHAPS and CHAPSO, the process was shown to fit
well to three-stage model previously proposed for other
detergents . According to the structural characteristics
of CHAPS, we can divide its states in aqueous solutions
into three parts for purple membrane solubilization [23,24].
(1) 0–4 mM: It is under the CMC of CHAPS. Because the
tail of CHAPS is inserted into the inner area of the bilayer
of purple membrane, a slight disturbance takes place in
lipid bilayer without destroying the integrity of the bilayer.
The high viscosity in these area of purple membrane
bilayer is formed. The results show that rotational correla-
tion time increases gradually. The isomerization of retinal
Relationship between I
/I cm and CHAPS concentrations
CHAPS (mmol/l)02468 10 121416
I /I cm0.510.58 069 0.940.58 0590.55 0.540.54
K. Hu et al. / Journal of Photochemistry and Photobiology B: Biology 58 (2000) 163–169167
Fig. 6. ESR spectra of purple membrane labeled by 16-doxyl-stearic acid, free radical, treated with different concentrations of CHAPS.
is not easy and photochemical cycle becomes difficult. The
change of lipid environment of BR in PM results in
increasing of t
. (2) 4–10 mM: The range of
formation of CHAPS micelles, both CHAPS monomers
and micelles exist in equilibrium. Lipids are removed by
CHAPS, and some proteins are solubilized. The integrity
of the lipid bilayer is damaged, so the environment of BR
greatly changes. The t reached the maximum at 5 mM
CHAPS concentration. It results in the sharp increase of
near 4 mM CHAPS treatment. (3) Above
10 mM: This is above the CMC of CHAPS. Lipids around
the membrane protein are exchanged for detergent, re-
sulting in formation of mixed CHAPS-lipid micelles,
CHAPS-protein micelles and CHAPS-lipid-protein mi-
celles. In the presence of excess CHAPS, micelles are in a
more stable state, so both t
The effects of CHAPS on the proton yield, M
4) show that when CHAPS concentration reaches 3 mM,
the proton yield is the lowest, the M
minimum, above 8 mM CHAPS both proton yield and
M tends saturate. These results also show the effect of
the lipid environment of BR on the proton pump function
and photocycle of BR. If the change of M
decay kinetics could be traced simultaneously, the under-
standing of relationship between them might be obtained.
Studying this problem should benefit the understanding of
the mechanism of the relation between the BR photocycle
and the proton pump. It was noticed, t
increases when CHAPS concentration increases from 0 to
10 mM.When above 10 mM, both of them tend to saturate,
but proton yield and Mhave the minimum at 3 mM, it
means the proton yield and M
directly with the t
From ESR experiments, it is noticed that between 10
and 20 mM CHAPS concentration, t decreases. It means
that when CHAPS concentration reaches 20 mM, the
purple membrane will be solubilized completely.
The ratios of Mto other intermediates show the
are not consistent
tend to saturate.
K. Hu et al. / Journal of Photochemistry and Photobiology B: Biology 58 (2000) 163–169
Fig. 7. Effect of CHAPS on rotational correlation time t of hydrophobic inner area in purple membrane bilayer.
relative quantity of M
reported that after treatment of CHAPS, the kinetics of
M of BR show an increase in the rise time and much
larger increase in the decay time . Therefore, it is
reasonable that the ratios of M
increase when the BR is treated by CHAPS. From our
experiments, the M decay increases obviously from 2
mM CHAPS concentration, the ratios of M
intermediates also increase from 2 mM CHAPS con-
centration. The M decay sharply increases from 2 to 6
mM CHAPS concentration and the relative quantity of
Min the photocycle reaches the maximum near 6 mM
CHAPS. After 10 mM CHAPS concentration, both the
M decay and ratios of M
The study of the effect of environment on the structure
shows detergent solubilization results in changing the
protein conformation . Upon partial delipidation or
change occurs . The change of absorption maximum of
BR (Fig. 1) also means the change of protein structure. In
this work we have investigated more thoroughly the
profound influence on BR photocycle, proton pump func-
tion and membrane viscosity by treatment of purple
membrane with different CHAPS concentrations. We sug-
gest CHAPS affects the lipid environment of BR, the effect
of environment results in the conformation change of BR.
The change of conformation leads the change of photocy-
cle and proton pump function of BR.
Some other detergents, such as Triton X-100, and
melittin that affect the lipid environment of BR also lead
the change of photocycle and proton pump function of BR
[26–29]. These studies show the importance of maintain-
ing a native lipid environment of BR.
in the photocycle of BR. It was
1. Further confirm the association between M
proton pumping function.
2. In the range of its critical micelle concentration (CMC),
CHAPS makes largest effect on M
decay of BR.
3. The proton yield and M
concentration reaches 3 mM. Largest effect of CHAPS
on proton yield and M
micelle concentration. They are not consistent with the
M decay and proton decay.
4. CHAPS makes the changes of rotational correlation
time (t ) of hydrophobic inner area in PM and ratios of
M to other intermediates. The t reaches the maxi-
mum at 5 mM CHAPS concentration, and the ratios
reache the largest at 6 mM CHAPS concentration. Both
of the largest effects occur in the range of CMC.
5. The studies of the effect of lipid environment in purple
membrane show the importance of maintaining a native
lipid environment of BR.
to other intermediates
decay and proton
are lowest when CHAPS
occurs before its critical
to other intermediates tend
Means purple membrane
Means rotational correlation time
Means the half time of M
Means the half time of proton decay
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