Tryptophan fluorescence quenching by alkaline earth metal cations ...

Journal of Photochemistry and Photobiology B: Biology 59 (2000) 38–41
www.elsevier.nl/locate/jphotobiol
Tryptophan fluorescence quenching by alkaline earth metal cations in
deionized bacteriorhodopsin
Guangyu Wang, Ao-Jin Wang, Kun-Sheng Hu*
Institute of Biophysics, Academia Sinica, Beijing 100101, PR China
Received 14 July 2000; accepted 3 November 2000
Abstract
Tryptophan quenching by the addition of alkaline earth metal cations to deionized bacteriorhodopsin suspensions was determined. The
results show that the addition of cation primarily quenches fluorescence from surface tryptophan residues. The quenched intensity exhibits
a1/R dependence, where R is the ionic radius of the corresponding metal ion. This observation results from a stronger energy transfer
coupling between the tryptophan and the retinal. The membrane curvature may be involved as a result of cations motion and correlated
conformational changes. © 2000 Elsevier Science B.V. All rights reserved.
Keywords: Bacteriorhodopsin; Fluorescence quenching; Energy transfer coupling
1. Introduction Trp residues [9,13,14]. Protein fluorescence studies
showed that deionization causes an increase in emission
Bacteriorhodopsin (bR) is a retinal-containing protein in intensity and a small bathochromic shift. The difference
the purple membrane of Halobacterium halobium [1]. spectrum contains primarily emission at 333 nm, which
Upon illumination, the protein undergoes a photocycle and could reflect the emission of surface tryptophan residues
pumps protons across the cell membrane. The cumulative [15,16]. The quenching of the emission at 333 nm by all
proton gradient drives ATP synthesis [2–4].
metal cations was interpreted in terms of an energy transfer
The bR protein contains eight tryptophan (Trp) and 11 coupling between tryptophans and retinal [17]. UV differtyrosine
residues [5]. It has been proposed that all but one
21
ence spectra of the blue membrane upon Mn binding
or two Trp residues are involved in an energy transfer indicated a charge perturbation of Trp residues [14]. The
between the retinal chromophore and the Trp residues. neighboring charged species possibly involve a divalent
Surface Trp residues such as Trp10, Trp12 or Trp137 were metal cation [18]. In this study alkaline earth metal cations
recently specified as the candidates for the unquenched or were employed to quench Trp fluorescence in the deionpartly
quenched Trp residues [6].
ized bR suspensions. Our results show that the addition of
21 21
The protein contains 4 mol of Ca and 1 mol of Mg cation primarily quenches fluorescence from surface Trp
per mol of bR [7]. At neutral pH removal of these cations residues. The quenched fluorescence intensity exhibits a
leads to a purple-to-blue color transition and eliminates 1/R dependence, where R is the crystal ionic radius of the
proton pumping by inhibiting the Schiff base deprotonation corresponding metal cation. The feature could be explained
[7,8]. On the other hand, the spectroscopic and photo- by the membrane curvature which causes a change in
chemical properties of the protein can be restored by energy transfer between retinal and Trp residues.
adding divalent cations to the deionized blue membrane
suspensions [9–12].
Several groups have demonstrated that removal of metal 2. Materials and methods
cations causes a small conformational change around the
Purple membrane was prepared from Halobacterium
halobium strain RM
1 1
as previously described [19]. The
*Corresponding author. Present address: Institute of Biophysics, 15
Datun Road, Beijing 100101, PR China. Tel.: 186-10-6488-8580; fax: samples was then deionized passing purple membrane
186-10-6487-7837.
suspensions through a cation exchange column (Dowex
E-mail address: huks1401@sun5.ibp.ac.cn (K.-S. Hu). 50W). Reionization was carried out by adding MgCl
2,
1011-1344/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved.
PII: S1011-1344(00)00132-9

G. Wang et al. / Journal of Photochemistry and Photobiology B: Biology 59 (2000) 38 –41 39
CaCl
2, SrCl2 and BaCl2
to the deionized bR samples until
no further spectral change could be detected. All the
samples were light-adapted and measured at 5 mM based
21 21
on that ´
605
of the deionized bR is 60 000 M cm and
21 21
´
568
of the native bR is 63 000 M cm , where ´ is the
molar extinction coefficient [9].
Absorption spectra were measured on a Hitachi U-3200
UV/Visible spectrometer and steady state fluorescence
spectra on a Hitachi F4500 spectrometer, where the
excitation wavelength was set at 28065 nm. All the
spectra were recorded at pH 5.5 and 258C.
3. Results
Fig. 1a shows the steady-state fluorescence emission
spectra of bR. The bR emission band has a maximum at
319 nm. The deionized bR gives a peak at 325 nm and its
Fig. 2. Titration curves of the bR emission change as a function of added
21 21 21 21
divalent cations. j: Mg . d: Ca . m: Sr . .: Ba . The bR
concentration is 5 mM. pH55.5.
fluorescence intensity is about 30% higher than that of the
21 21 21 21
native sample. When four Mg , Ca , Sr or Ba per
bR are added to the deionized bR suspension, most of the
fluorescence is quenched (Fig. 2). The necessary salt
concentration to level off is about 12 cations per bR.
Fig. 1b shows Trp quenching by different alkaline earth
metal cations at concentration higher than 12 ions per bR.
The difference spectrum of bR minus the deionized bR is a
control. It is interesting that the quenched emission intensi-
21 21 21
ty is different. The effect order is Mg .Ca .Sr .
21
Ba . However, the shapes of the difference spectra are
similar to that of the difference between bR and DbR.
They all have a minimum at 333 nm. The emission around
this wavelength could result from the fluorescence of a
surface Trp residue [16,17].
Compared to the emission of native bR, relative quenching
efficiencies of four kinds of metal cations in deionized
bR are obtained. Fig. 3 shows that they are almost a linear
function of wavelength from 300 to 390 nm. This suggests
that the binding of cations in the deionized bR is similar to
that in the native bR. Additionally, the decreasing order of
21 21 21
relative quenching efficiency is still Mg , Ca , Sr and
21
Ba . This is contrary to a change in their size [20]. When
21 21 21
relative quenching efficiencies of Ca , Sr and Ba
ions are multiplied by their size and then divided by
21
relative quenching efficiency of Mg multiplied by its
size, three ratios of one are obtained between 300 and 390
21
nm within an error. Fig. 4 reveals a case about Ca . The
others are very similar. This means that the quenched
Fig. 1. (a) Steady state Trp fluorescence spectra of bR suspended in emission intensity depends on 1/R, where R is the ionic
water. Successive spectra with decreasing amplitude correspond to the
21 21 21
deionized bR, Ba -regenerated bR, Sr -regenerated bR, Ca -regener- radius of the corresponding metal ion.
21
ated bR, Mg -regenerated bR, and native bR. The bR concentration is 5
mM. pH55.5. The samples were excited at 28065 nm. (b) Fluorescence
spectral change of deionized bR upon the addition of cations at con- 4. Discussion
centration higher than 12 cations per bR. Successive spectra with
21 21 21 21
increasing amplitude correspond to Ba , Sr , Ca and Mg in the
sample cuvette containing the DbR suspensions. The curve with the
largest amplitude is the difference spectrum between native bR and
deionized bR. The bR concentration is 5 mM. pH55.5.
In this study all the mission spectra were recorded at a
bR concentration of 5 mM, pH 5.5 and 258C. Under this
conditions, the emitted light is only slightly reabsorbed by

40 G. Wang et al. / Journal of Photochemistry and Photobiology B: Biology 59 (2000) 38 –41
ther indicated the conformational change perturbed the
21
tryptophan [14]. In addition, the addition of Ca to the
ion-depleted and retinal-removed membrane does not
affect fluorescence emission intensity [17]. These results
suggest the quenching of the fluorescence emission by
alkaline earth metal cations is due to a stronger energy
transfer coupling between the tryptophan and the retinal.
Different energy transfer efficiencies may be due to a
difference in their relative distance or orientation to retinal.
It has been reported that the affinity of divalent metal
cations changes with their sizes. The smaller ions are more
tightly bound to the membrane than larger ones [21,22].
The different affinity would result in a different extent of
13
conformational change. On the other hand, C NMR
studies show that the bound cations exchange rather
rapidly among various types of cation binding sites [23].
Fig. 3. Emission wavelength dependence of relative quenching efficiency
Varo ´´ et al. also proposed that cations bind to a non-specific
(Q) of the metal cations at concentration higher than 12 cation/bR.
Successive curves with increasing amplitude at 333 nm correspond to site [24]. Therefore, cation motions and correlated con-
21 21 21 21
Ba , Sr , Ca and Mg in the sample cuvette containing the formational changes in the bR result in transient forces.
deionized bR suspension. The bR concentration is 5 mM. pH55.5. These transient forces change the membrane curvature
Relative quenching efficiency is calculated as Q 5 uIi2 Ibmu/uIbm2 Ipm u, [25]. Because of different affinities of divalent cations on
where I
bm, Ipm and Ii are relative emission intensity of deionized bR,
the membrane, the membrane curvature would be different.
native bR and cation-regenerated sample, respectively.
The energy transfer efficiency is thus changed due to
different distance between tryptophan and retinal and their
ground-state bR molecules and the emission spectra that
relative orientation.
we recorded are not distorted by any internal filter effect,
even by the high bR concentration [16]. In addition, at this
concentration, the deionized bR samples are not easily
reionized by minute traces of any cation and extreme care
5. Abbreviations
has not to be taken in order to keep a completely deionized
suspension even for very short periods of time. What is
bR Means bacteriorhodopsin
more, the use of a higher bR concentration could help us
Trp Means tryptophan
get around the problem that the Raman emission from
DbR Means deionized bacteriorhodopsin
water at 298 nm induces a noticeable shoulder on the
fluorescence band which makes measurements at wavelengths
shorter than 310 nm questionable [16].
Acknowledgements
The circular dichroism studies show that the addition of
cation to the deionized blue membrane changes the protein This work was funded by National Science Foundation
conformation [9,13]. UV-difference spectrophotometry fur- of China and Grant for Key Program from Chinese
Academy of Sciences (Grant Nos. Kj951-A1-501-05 and
Kj952-S1-03).
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