- Email: [email protected]
The thermal stability of native, delipidated, deionized and regenerated bacteriorhodopsin
Journal of Photochemistry and Photobiology, B: Biology, 7 (1990) 289-302
289
THE THERMAL STABILITY OF NATIVE, DELIPIDATED, DEIONIZED AND REGENERATED BACTERIORHODOPSIN G. C. KRESHECK,C. T. LIN*, L. N. WILLIAMSONand W. R. MASON Depa~ment of Chemistry, Northern Illinois University, DeIfalb, IL 50115 (U.S.A.)
D.-J. JANG and M. A. EL-SAYED Depa.,~.,~nt of Chemistry and Biochemistry, University of Californio~ Los Angeles, CA 90024 (U.S.A.)
(Received October 16, 1989; accepted January 25, 1990)
K e y w o r d s . Thermal stability, Bacteriorhodopsin, deprotonation.
Summary Differential scanning calorimetry curves and circular dichroism spectra were determined for native bacteriorhodopsin (bR), deionized bR, acid blue and acid purple bR, 75% delipidated bR, deionized-delipidated bR and Mg~+ regenerated deionized bR. The effects of the different perturbations on the thermal stability (melting temperature) and the apparent helical content of the protein were examined. These perturbations have more influence on the deprotonation efficiency and the color change of retinal than on the thermal stability and the apparent helical content of the protein. Although the addition of Mg 2+ to deionized bR restores the photochemical cycle, it does not restore the thermal stability to that of the native material.
I. Introduction
The retinylidene protein bacteriorhodopsin ('oR), the other photosynthetic system besides chlorophyll, is one of the protein pigments found in the purple membrane of I - I a I o b c w t e r i u m h a I o b i u m [1, 2]. Light-adapted bR contains an all-trans retinal which is covalently bound to the protein via a protonated Schiff base (PSB) [1, 3]. On absorption of visible light, bR undergoes the photochemical cycle [4] h~,
bR56s ps ~ Ke10 ~
L550
--H + 60 ~ ' M412
+H + ms ' Oe40
'
bR56s
AS a result, the PSB is deprotonated during the L550-* M412 trzlisition, leading to a proton pumping process that increases the proton concentration on the ~Author to whom correspondence should be addressed. Elsevier Sequoia/Printed in The Netherlands
290 outside surface of the membrane [5]. The proton gradient created across the membrane is then used to transform ADP into ATP in the final step of photosynthesis [6]. Understanding the molecular mechanism of deprotonation of the PSB thus becomes very important to our understanding of the molecular mechanism of solar energy storage in nature. Recently, the effects of various perturbations on the probability of the deprotonation process have been studied in detail by a number of research groups. Acidification (to a pH below 3) or removal of cations from bR produces a blue form o f b R [7-10]. This form is incapable of deprotonating to form M41e [11], but still allows the isomerization of retinal and the formation of the IQ]o and L55o analogues [11, 12] to take place. The purple color of bR returns on further acidification of acid blue bR; however, although acid purple bR has K610 and L550 analogues, it does not form M41e nor does it pump protons [11]. Changing the concentration of metal cations in bR is found to change the deprotonation probability non-linearly, but does not change the relative population of metal cation (i.e. Eu 3+) sites [13]. This suggests an indirect involvement of metal cations in the deprotonation process. Corcoran et al. [ 13 ] have observed that most of the luminescence of the bound Eu 8+ originates from surface sites. This observation suggests an indirect involvement of surface metal cations in the control of the protein conformational changes, which have previously been proposed [14, 15] as a mechanism for controlling the rate of the deprotonation process. Following retinal absorption [16, 17] and deprotonation probability of the PSB [18], the effect of removing 7596 of the bR lipids (to form delipidated bR) has been studied as a function of pH and metal cation concentration. The results of these studies provide further indirect evidence of the importance of surface charges in controlling the protein conformation (and thus the retinal color) [16, 17] and protein conformational changes during the photochemical cycle (and thus the deprotonation rate) [14, 15, 18]. The above discussion strongly indicates that protein structure plays a dominant role in the mechanism of the deprotonation process. It has previously been shown [ 19-21 ] that a thermodynamic analysis of the structural stability of bR can be obtained from studies of the thermal unfolding by differential scanning calorimetry (DSC). Previous circular dichroism (CD) studies of bR in various regions of the spectrum have also been used to monitor changes in the environment of the retinal chromophore (visible), the aromatic amino acids (near UV) and the secondary structure of the protein (far UV). These studies have indicated that there are no changes in secondary structure between pH 2.4 and pH 11.8 [22], and that the far-UV CD spectra of lightand dark-adapted bR in solution are identical [23] as are the near-UV CD spectra in acrylamide gels at pH 4 and 7 [8]. The far-UV CD spectra of native and dellpidated oriented films of purple membrane at 15°/0--20% relative humidity have recently been reported to be almost superimposable [24]. In this work, the effect of several different perturbations on the overall macroscopic protein is studied by DSC and CD spectrometry. A comparison is
291 then made between the response to these different perturbations of the protein stability and helical content on the one hand and the structure and function of the active site (as measured by the changes in the deprotonation efficiency) on the other.
2. Experimental details The procedure used for the growth and purification of the purple membrane was a combination of the methods outlined by Oesterhelt and Stoeckenius [25] and Becher and Cassim [26]. The deionized bR samples were prepared by flowing bE through a cation exchange column and washing several times with double deionized water. The delipidated bR was prepared by incubating 10 mg of purple membrane sample overnight at room temperature in 5 ml of 20 mM 3-[(3-cholaxnidopropyl)dimethylanu~onio]-l-propanesulfonate (CHAPS; Pierce Chemical Co., Rockford, IL) containing 5 m_M acetate buffer CpH 5.4) [16]. Deionized distilled water was used to prepare all solutions, and samples were used promptly or stored in polyethylene bottles at 4 °C. Unbuffered bR suspensions which were not deionized gave a measured pH of 5 . 5 ± 0 . 5 , whereas the pH of the deionized samples was slightly lower, e.g. 4 . 0 ± 0.5. These solutions were used for DSC and CD studies without additional buffering components in order to provide the same conditions as used in previous photochemical investigations [18]. Protein concentration was determined from the optical absorption at 280 nm using an extinction coefficient of 113 400 M -1 cm -1. The concentration of delipidated samples was corrected for the small absorbance change (7%) that accompanies removal of phospholipid from purple membrane [24]. The absorbance at 222 nm was also used to corroborate the concentration for all protein solutions. The CD spectra were determined in ambient conditions, 22 ± 1 °C, using a computer-controlled spectrometer built at Northern Illinois University and described elsewhere [27]. The path length of the quartz cells used for the CD measurements was 1.00 cm and the CD scale was calibrated using camphor sulfuric acid solutions [28--30]. The molar ellipticity was calculated using a mean residue weight of 105. The observed differential absorbance ranged from approximately 5 × 1 0 -5 to 1 . 6 × 1 0 -a with an average noise level of 5 × 10 -~ from 190 to 430 nm. For the determination of the melting point by observing color changes from purple (or blue) to yellow, the pH was maintained between pH 3 and 7 with 0.1 M acetate buffer, but was adjusted with HC1 below pH 3. All solutions of native bR for which the pH was 2.5 or below exhibited visual evidence of aggregation. A spectrophotometric method was also used to monitor the effect of temperature on the disappearance of the visible absorption band of bE. The results were the same as those obtained from the visual "melting point" observation. The calorimetric studies were carried out using an MC-1 differential scanning calorimeter (Micro Cal, North Hampton, MA) which was interfaced
292 to an IBM 9000 microcomputer system as previously described [31 ]. Sample and solvent reference solutions of 0.61 ml were added to matched stainless steel cells (Hastelloy C-276) and data were collected at a heating rate of 1.088 K min -1 except for a few runs at a heating rate of 0.357 K min -1. The transition temperature tm (°C) or Tm (K) corresponds to the temperature of maximum excess heat capacity for the transition. No dependence of tm on the scanning rate was noted for any of the samples studied. The total enthalpy change Ah (cal g-1) was found from the area under the peak using an electrical pulse to convert the observed area to units of calories. Numerical integration of the peaks was performed using linear baselines connecting points just below and above the temperature observed for the transitions. The molar calorimetric enthalpy change h/-/¢ was obtained as the product of Ah and the molecular weight of bR (27 000 g mol-1). The van't Hoff enthalpy change AHv~ was estimated assuming a two-state process from the equation [32 ] ~-tvH ----- 4RTm2Cpm/Ah
(1)
where Cp~. is the maximum excess specific heat observed at t~. In most cases, the data above t~ were not symmetrical with respect to the data below tm due to deviations from simple two-state behavior. No attempt was made to perform additional curve fitting due to the unknown state of association of the protein before and after thermal denaturation.
3. R e s u l t s
The temperatures at which the native bR solutions turned yellow in the pH range 0.56-6.50 are given in Fig. 1. This temperature is found to correlate with the transition temperature for the thermal denaturation of the protein, at least in the pH range 5 - 6 used in our DSC studies. The decrease in apparent stability of bR with decreasing pH, as shown by a lower tin, is not unexpected for proteins [33]. The possible loss of metal ions associated with the chromophore and/or the protonation of acidic residues near pH 3.0 are indicated by the blue color of the unheated solutions. The apparent increase in stability below pH 1.5, indicated by an increase in tin, is unexpected. The bR solutions in this pH region are again purple in color at room temperature, but have a CD spectrum very similar to heat-denatured protein. There are at least two possible sources that might account for the increase in the thermal stability of acid purple bR: (i) the protonation of the phospholipid head groups (pKa < 2.5) takes place leading to a change in secondary structure; (ii) the aggregation affects the surface amino acids. The fact that no enhancement in the thermal stability is observed in Fig. 1 for acid blue bR (pH 2.5), where aggregation is also apparent, suggests the importance of secondary structure change in the thermal stability of bR. The alteration of the active site in acid purple bR is such that the isomerization of retinal and formation of Kelo and L55o occur, but deprotonation does not take place.
293
100
90
80
tm(°C) 70
60
50
'
42
2.8
5.6
7.0
pH Fig. 1. Plot of the melting t e m p e r a t u r e tm vs. pH for native bR b a s e d o n the appearance of a yellow solution. The line drawn is empirical.
3.1. D S C s t u d i e s The thermal stabilities of native, delipidated and deionized-delipidated bR were investigated, and endotherms were observed with tm in the range 9 0 - 1 0 0 °C (the b o t t o m three curves of Fig. 2) where the solutions changed color from purple to yellow. All three curves were asymmetrical, with the suggestion of a possible exothermic step above the midpoint t e m p e r a t u r e for the transitions that could result from protein association effects [34]. The thermal denaturation of bR was found to be irreversible from the absence of any transition for samples which were cooled to r o o m t e m p e r a t u r e on completion of the DSC scans and rerun. A summary of the t herm odynam i c data is given in the first four columns of Table 1. The data were analyzed by assuming a single two-step protein denaturation mechanism. By fitting the data up to the t em perat ure of m a x i m u m heat capacity, using the p r o c e d u r e of Kresheck and E rm an [35], the theoretical curves (broken lines) in Fig. 2 were obtained; the differences bet w een the observed and calculated curves were also plotted along the linear baseline drawn between the t e m p e r a t u r e just below and above the transition temperature. The deviations of the residuals from the linear baseline up to tm are comparable with the noise level of the baseline. The sharp decrease in the residuals below the linear baseline above tm reflects the effect of the possible exothermic step indicated above in addition to deviations from the assumed two-step mechanism. The values of AHc and AHvn for representative runs are given in the fifth and sixth columns of Table 1 for the native, delipidated, deiordzed-delipidated, deionized and regenerat ed samples. The van't Hoff enthalpy change is about 33% greater than the calorimetric enthalpy change for the native protein and more than twice as large for the delipidated
294
Delipidated
pH 25
8:1 Mg2+ >}< Q. < O I..< LU "1" (,9 o0 LU O X LU
4:1 Mg2+
Deionized
Deionized- . ~ delipidated
Delipidated
,f~--m~- f~
~,~_
/
~
',
---~-~~'~-'~ j/r~,
Native
2'0
4'0
6'0
8'0
~~0
TEMP (DEG°C) Fig. 2. DSC curves for the thermal denaturation of bR in the native (4.68 g 1-1), delipidated (4.68 g l - l ) , deionized-deUpidated (4.68 g 1-1), deionized (3.14 g l - l ) , regenerated (4:1 Mg~+ (3.14 g 1-1); 8:1 Mg~+ (3.07 g 1-1)) and acid delipidated (pH 2.5) (2.34 g 1-1) states. Each division on the ordinate corresponds to 2.87 mcal K -1 at a scanning rate of 1.088 K rain -~. The calibration pulses around 45 °C for the deionized-delipidated samples correspond to 11.15 mcal. The smooth broken line that follows the sample was calculated according to the procedure described in the text. The linear b r o k e n baselines were used as reference lines for plotting the residuals between the observed and theoretical curves.
samples. The presence of intermolecular steps in the denaturation process is indicated when AHvH exceeds AH c [36]. It is not within the scope of this study to address this question in more detail due to the unknown state of association of the samples, which are known from the literature to be suspensions rather than true solutions. The thermal stabilities of deionized samples of bR was investigated, and results for a representative sample are given in Fig. 2 (fifth curve from the top). Three major differences between the deionized and native samples may
295
c~ i
0
0
0 0 cO
O0 00 ~Ob-
cO
I
2g 00
I
•~
I
I
t
I
1
0
0 c~
0
0
~
-e i
0
I
c~ O o
c~
Lo
¢o
~oo~
c~ e~
0
A
°
~
Oc~oO
P, 0
/
296 be noted. The peak for the deionized sample is smaller, m ore symmetrical and occurs at a lower t em pe r at ur e than the peak for the native sample. In addition, attempts were made to regenerate the native form of bR starting with the deionized state by restoring Mg 2÷ ions. The DSC spect ra obtained are given in Fig. 2 (second and third curves from the top) for 8:1 and 4:1 Mg 2+ respectively, for samples which were regenerat ed and aged 24 h at 4 °C before analysis. It should be not ed that these curves in the middle of the figure are amplified 2.5 times, i.e. the two deionized curves in the middle of the figure are the same e x c e p t for the scale difference. The addition of 4:1 Mg 2÷ restores the original purple color and photocycle of bR but only increases tm slightly. The values of AHc and AHv~ obtained for these samples are also given in the fifth and sixth columns of Table 1. The curve at the top of Fig. 2 represents the scan obtained for a delipidated sample that was adjusted to pH 2.5 (scale amplified 2 times). This sample was purple in color and showed no signs of phase separation, in contrast with the native protein which turns blue and precipitates. The delipidated sample also gave data with a good signal-to-noise ratio, and exhibited a small narrow peak around 90 °C. Under the same conditions, native bR showed a very ragged trace with no sign of a thermal transition within our experimental sensitivity setting. 3.2. CD s p ectr a The CD spectra of native, thermal denatured, acid blue (pH 2.5), acid purple (pH 0), deionized and regenerated samples were determined in order to obtain an indication of the changes in the secondary structure for the different samples. The results from these studies are given in the Iast column of Table 1, together with reference data for [0]222 [37] for the a helix, fl form, fl turns and u n o r d e r e d state as they occur in typical globular proteins. for bR, there is compelling evidence [38] that some of the a helices may be in the an form, rather than exclusively in the a~ form. The an form, due to the tilting of its amide plane relative to the helical axis, exhibits much less ellipticity for an identical helical content than the a~ form in which the amide plane is parallel to the helical axis. As expected, the value of [01222 for the native form of bR indicates the pr esence of a considerable a-helical content. No significant difference in s e c onda ry structure is suggested for the native, deionized, delipidated and r egener a t ed samples. Both the heat- and acid-treated samples appear to have lost a portion of their a-helical content, and the values of [0122z are less than the value of - 13 800 e x p e c t e d for an unor de r ed sample. It is important to note that [01222 for the u n o r d e r e d form in proteins varies over a very wide range of values. The value - 13 800 referred to in Table 1 was obtained by averaging the values for 15 extrinsic proteins. When the values for only five of the 15 extrinsic proteins were used, an average value of about + 1580 was obtained [39]. The effects of aggregation on the CD measurements of bR at pH 0 and 2.5 are uncertain. Figure 3 shows the CD (top) and absorption (bottom)
297 1.0
10 s/kA @.@
I
2@0.@
I
[email protected]
240.@
(rim)
ABS
I.8 1 ~ s B .5
@.0
.
~ i 2B@. I~
i 22B. B
i 24@. @
~k.Cnm) Fig. 3. CD (top) and absorption (bottom) spectra of 5.2x10 -7 M bR in water: (1) native purple bR; (2) heat-treated (or denatured) yellow bR; (3) acid blue bR (pH 2.5); (4) acid purple bR (pH 0); (5) deionized blue bR. s p e c t r a of 5 . 2 x 1 0 - ? M bR in water: (1) native, (2) t h e r m a l l y d e n a t u r e d , (3) acid blue (pH 2.5), (4) acid p u r p l e (pH 0) a n d (5) deionized. Several o b s e r v a t i o n s can b e s u m m a r i z e d . (i) As m e n t i o n e d in S e c t i o n 2, all solutions for w h i c h the p H is 2.5 a n d b e l o w exhibit visual e v i d e n c e o f aggregation. H o w e v e r , at this low c o n c e n t r a t i o n o f 5 . 2 x 1 0 -7 M, all five s a m p l e s s e e m to s h o w a h o m o g e n e o u s solution with n o visible p a r t i c u l a t e s u s p e n s i o n . (ii) The a b s o r b a n c e at ; t - - 2 2 2 n m for all five s a m p l e s is r o u g h l y the same, i . e . A -- 0.5. At A----222 nm, the AA value for e a c h o f the five s a m p l e s (top p o r t i o n o f Fig. 3) is quite distinct. T h e v a l u e s o f [e]222 are listed in Table 1.
298 (iii) In the b o t t o m spectra of Fig. 3, curves 3 and 4 for bR at acidic pH show greater absorbance in the region above 230 nm and lower absorbance around 200 nm. The former may reflect the greater scattering as a result of aggregation and the latter may be due to the Duysens absorption flattening effect which is a concentration obscuring phenomenon. (iv) In the top spectra of Fig. 3, a 70% reduction in ellipticity at maximum absorbance wavelength (A = 200 nm) is observed for acid blue and acid purple hR. Curves 3 and 4 show a depression of the 208 nm CD band and a change in cross-over wavelengths to longer wavelengths. The effect is greater at pH 0 than at pH 2.5 due to differential Mie light scattering. (v) Curve 2 in Fig. 3 shows an approximate 109/0 increase in absorption and 30% decrease in ellipticity at the extreme, indicating some conformational changes for the heat-treated samples. Observations (iii) and (iv) are quite similar to the changes observed in the absorption and CD spectra due to a pH change from 3.85 to 2.4 in helical polyglutamic acid suspensions [40]. Urry [40] has described a pr oc e dur e for estimating the molar ellipticity at 222 nm of polypeptides that exist as suspensions rather than molecularly dispersed solutions. He notes the following at 222 urn: (a) the effects due to differential scattering of left and right circularly polarized light are zero; (b) the absorbance of a sample adequate to penetrate the UV to 192 um would be low, such that the Duysens absorption flattening coefficient would be close to unity; (c) the absorbance at 222 nm exhibits little h y p e r c h r o m i s m or hypochromism on changing protein conformation. In the light of these facts, observation (ii) and the results in Fig. 1, it is suggested that the aggregation is not the only factor responsible for the [0]222 value observed in acid blue and acid purple bR.
4. D i s c u s s i o n a n d c o n c l u s i o n s The [ 0]222 value obtained for native bR ( - 15 600 :i=600 deg cm 2 d m o l - 1) compares well with the value of - 15 900 deg cm 2 d m o l - 1 at 223 nm r e p o r t e d by Becher and Cassim [41]. In addition, our observation that delipidation does not significantly alter the value of [0]222 is in accord with the findings of Hartsel and Cassim [24], although Triton X-100 was used for lipid removal in their work and CHAPS in ours. However, we do not concur with the hterature with r es pe c t to the effects of acidification to pH 2 . 4 - 2 . 5 on [ 0]222. Muccio and Cassim [22] r e por t ed that this value is unchanged between pH 2.4 and 5.0, whereas our value decreased from - 1 5 600 to - 9 1 0 0 deg cm 2 dmo1-1 with a decrease in pH from 5.5 :fi0.5 to 2.5. In spite of the aggregation observed in the acid blue and acid purple bR samples, it is difficult to explain this discrepancy since Muccio and Cassim not ed that optical artifacts due to absorption flattening, light scattering and the differential hght-scattering effect of circularly polarized light are minimal at 222 rim.
299 However, our results at 222 nm (which reflect the contribution of the peptide backbone) parallel the results of Mowery et al. [8] at 315 a m (which reflects the contribution of the retinal chromophore), where the pH 2.0 spectrum showed a marked decrease in ellipticity at 315 nm when compared with the ellipticity at pH 4 or 7. Our results thus suggest a possible protein conformational contribution to other known changes in physical and chemical behavior of bR between pH 0 and pH 2.5 [8]. The thermodynamic data (tin, AHc and AHvu) obtained in this study for native bR in water are in good agreement with the results obtained by previous workers [19-21]. Our value of tm (97.1 ± 1.4 °C) for the "100 °C" transition is closest to that of Jackson and Sturtevant [19] ( 9 5 . 0 ± 0 . 7 °C), but our AH¢ value (92 ± 3 kcal mol-1) is closest to that reported by TristramNagle et al. [20] (91___9 kcal mol-~). It is noted that the AH¢ value given is an average of five runs, whereas those reported in Table 1 are representative values calculated from a single scan. Jackson and Sturtevant [ 19 ] found that the AHvH/AH¢ ratio was slightly less than 2 regardless of ionic conditions, and Brouillette et al. [21 ] obtained an average value of 1.9 from data collected between pH 6 and pH 11. The value we obtained for native bR in this study was also 1.9, again suggesting possible intermolecular cooperativity in the thermal denaturation of bR. The so-called "80 °C" pretransition was only evident in our most concentrated sample (5.8 mg m1-1) and it was located at 78.4 °C with a calorimetric enthalpy change of 9.7 kcal mol-x. This compares well with a temperature of 79.4 ± 0.8 °C and 6.50 ± 0.63 kcal m o l - 1 in water reported by Jackson and Sturtevant [19] for tm and AH¢ respectively for this transition. It is of interest that a purple to reddish-purple color change was observed visually at about 70 °C, which corresponds to the onset of this "pretransition". The significance of this color change is not known, but it could be due to the loss of crystallim'ty of the bR lattice which occurs at about 80 °C as reported by Hiraki et al. [42]. The effect of increasing the hydrogen ion concentration is first to decrease the melting point from 97 °C in bR until acid blue bR is formed (melting point = 65 °C). A further decrease in pH leads to an increase in the melting point as acid purple bR is formed (melting point above 90 °C; similar to native bR). Although acid purple bR resembles native bR in having a similar absorption maximum, similar melting point and similar initial steps in the photocycle (isomerization of retinal [ 12] and the formation of K82o and L55o [ 11 ]), acid purple bR has far less ~-helix content than native bR. Furthermore, the PSB in acid purple bR does not deprotonate during the cycle. This suggests that the deprotonation process, unlike the isomerization or melting temperature, is more sensitive to the conformational changes of the protein than the formation of the K~lo or Lsso forms during the cycle. This is consistent with the previous conclusion [ 13, 18 ] that the protonation process is controlled by the nature of the protein conformational change during the L550-~M4~ process (in contrast with the intra-retinal isomerization which occurs during the b R - ) K61o~ Lsso processes).
300 T h e a b o v e o b s e r v a t i o n s s u g g e s t t h a t the t h e r m a l stability of the p r o t e i n d o e s n o t c o r r e l a t e with t h e d e p r o t o n a t i o n ability o f the PSB. T h e d e n a t u r a t i o n t r a n s i t i o n t e m p e r a t u r e s o f native bR a n d d e l i p i d a t e d bR a r e f o u n d to b e similar, b u t t h e i r d e p r o t o n a t i o n efficiencies differ b y 50%. As e x p e c t e d , delipidation d o e s n o t c h a n g e the t h e r m a l stability or the a-helix c o n t e n t ( o r the s e c o n d a r y s t r u c t u r e ) o f t h e p r o t e i n at p H 5 . 5 ± 0 . 5 . H o w e v e r , it d o e s c h a n g e t h e solubility at p H 2.5 a n d t h e c h a r g e distribution [16, 17] w h i c h c a n h a v e effects on c o n f o r m a t i o n a l c h a n g e s d u r i n g the p h o t o c h e m i c a l cycle [18]. D e i o n i z a t i o n is f o u n d to d e c r e a s e the t h e r m a l stability o f t h e p r o t e i n as s h o w n in T a b l e 1. T h e t r a n s i t i o n t e m p e r a t u r e is a p p r o x i m a t e l y 70 °C, n o t far f r o m the acid b l u e bR m e l t i n g p o i n t ( a p p r o x i m a t e l y 65 °C) m e a s u r e d b y the visual t e c h n i q u e (Fig. 1). T h e e n t h a l p y o f d e n a t u r a t i o n o f deionized o r acid b l u e bR is o v e r 15% less t h a n t h a t for native bR. B o t h acid b l u e a n d deionized bR h a v e retinals t h a t i s o m e r i z e , b u t t h e i r PSB d o e s n o t d e p r o t o n a t e . T h e r e c o v e r y o f the d e p r o t o n a t i o n efficiency o f t h e PSB a n d t h e p u r p l e c o l o r o f bR o n addition o f c a t i o n s to d e i o n i z e d bR is f o u n d to b e r a p i d ( m i l l i s e c o n d s ) [43] a n d n o n - s p e c i f i c [10, 44].
Acknowledgments G. C. K. t h a n k s the NSF f o r s u p p o r t ( G r a n t No. P R M - 8 1 1 3 3 7 7 ) . C. T. L. w i s h e s to a c k n o w l e d g e the financial s u p p o r t f r o m t h e N o r t h e r n Illinois University G r a d u a t e S c h o o l a n d College o f Liberal Arts a n d Sciences. M. A. E. a n d D. J. J. wish to t h a n k the D e p a r t m e n t o f E n e r g y (Office o f B a s i c E n e r g y Sciences; G r a n t No. D E - F G 0 3 - 8 8 E R 1 3 8 2 8 ) f o r financial s u p p o r t .
References 1 D. Oesterhelt and W. Stoeckenius, Rhodopsin-like protein from the purple membrane of Halobacterium halobium, Nature ~ ) , New BIOL, 233 (1971) 149-152. 2 W. Stoeckenius and R. A. Bogomolni, Bacteriorhodopsin and related pigments of halobaeteria, Annu~ Rev. Biochem., 52 (1982) 587--619. 3 J. Bridgen and I. D. Walker, Photoreceptor protein from the purple membrane of Halobacterium hah~bium: molecular weight and retinal binding site, Biochemistry, 15 (1976) 792-798. 4 R. Lozier, R. A. Bogomolni and W. Stoeckenius, Bacteriorhodopsin: a light driven proton pump in Halobacterium halobium, Biophys. J., 15 (1975) 955--962. 5 N. Dencher and M. W'flms, Flash photometric experiments on the photochemical cycle of bacteriorhodopsin, Biophys. Struct. Mech., 1 (1975) 259-271. 6 J. W. Belliveau and J. K. Lanyi, Analogies between respiration and a light-driven proton pump as sources of energy for active glutamate transport in Halobacterium holobiur~ Arch. Biochem~ Biophys., 178 (1977) 308-314. 7 T. A. Moore, M. E. Edgerton, G. Parr, C. Greenwood and R. N. Perham, Studies of an acid-induced species of purple membrane from Halobacterium halobium, Biochem. J., 171 (1978) 469--476. 8 P. C. Mowery, R. H. Lozier, Q. Chae, Y.-W. Tseng, M. Taylor and W. Stoeckenins, Effect of acid pH on the absorption spectra and photoreactions of bacteriorhodopsin, Biochemistry, 18 (1979) 4100---4107.
301 9 Y. Kimura, A. Ikegami and V~. Stoeckenius, Salt and pH dependent changes of the purple membrane absorption spectrum, Photochern. Photobiol., 40 (1984) 641-646. 10 C. H. Chang, J.-G. Chen, R. Govindjee and T. Ebery, Cation binding by bacteriorhodopsin, Proc. Natl. Acad. Sci. USA, 82 (1985) 396-400. 11 E. L. Chronister, T. C. Corcoran, L. Song and M. A. E1-Sayed, On the molecular mechanisms of the Sehiff base deprotonation during the bacteriorhodopsin photocycle, Proc. Natl. Acad. Sci. USA, 83 (1986) 8580--8584. 12 T. Kobayashi, H. Ohtani, J. Iwai, A. Ikegami and H. Uehiki, Effect ofpH on the photoreaction cycles of bacteriorhodopsin, FEBS Lett., 162 (1983) 197-200. 13 T. C. Corcoran, K. Z. Ismail and M. A. E1-Sayed, Evidence for the involvement of more than one metal cation in the Schiff base deprotonation process during the photocycle of bacteriorhodopsin, Proc. Natl. Acad~ Sci. USA, 84 (1987) 4094-4098. 14 P. Dupuis, T. C. Corcoran and M. A. El-Sayed, Importance of bound cations to the tyrosine deprotonation during the photocycle of bacteriorhodopsin, Proc. Nat/. Acad. Sc/. USA, 82 (1985) 3662-3664. 15 J. H. Hanamoto, P. Dupuis and M. A. EI-Sayed, On the protein (tyrosine)--chromophore (protonated Schiff base) coupling in bacteriorhodopsin, Proc. Natl. Acad. Sci. US~ 81 (1984) 7083--7087. 16 I. Szundi and W. Stoeckenins, Effect of lipid surface changes on purple to blue transition of bacteriorhodopsin, Proc. Natl. Acad. Sci. USA, 84 (1987) 3681-3684. 17 I. Szundi and W. Stoeckenius, Purple-to-blue transition of bacteriorhodopsin in a neutral lipid environment, Biophys. J., 54 (1988) 227-232. 18 D. J. Jang and M. A. El-Sayed, Deprotonation of lipid-depleted bacteriorhodopsin, Proc. Natl. Acad. Sci. US~ 85 (1988) 5918--5922. 19 M. B. Jackson and J. N. Sturtevant, Phase transition of the purple membrane of Ha/obacterium, Biochemistry, 17 (1978) 911-915. 20 S. Tristram-Nagle, C. P. Yang and J. F. Nagle, Thermodynamic studies of purple membrane, Biochim. Biophys. Acta, 854 (1986) 58-66. 21 C. G. Brouillette, D. D. Muccio and T. K. Finney, pH dependence of bacteriorhodopsin thermal unfolding, Biochemistry, 26 (1987) 7431-7438. 22 D. D. Muccio and J. Y. Cassim, Interpretations of the effects of pH on the spectra of purple membrane, J. MoL Biol., 135 (1979) 595-609. 23 J. E. Draheim and J. Y. Cassim, Large scale global structural changes of the purple membrane during the photocycle, Biophys. J., 47 (1985) 497-507. 24 S. C. Hartsel and J. Y. Cassim, Structure and photodynamics of bacteriorhodopstn in a delipidated contracted lattice form of purple membrane, Biochemistry, 27 (1988) 3720-3724. 25 D. Oesterhelt and W. Stoeckenius, Isolation of the cell membrane of Halobacterium halobium and its fractionation into red and purple membrane, Methods Ene.~jmol., 31 (1974) 667-678. 26 B. M. Becher and J. Y. Cassim, Improved isolation procedures for the purple membrane of Halobacterium halobium, Prep. Biochern., 5 (1975) 161-178. 27 W. R. Mason, Spectrometer for simultaneous measurement of absorption and circular dichroism spectra, A n a l Chem., 54 (1982) 646-648. 28 P. H. Schippers and H. P. J. M. Dekkers, Direct determination of absolute circular dichroism data and calibration of commercial instruments, Anal. Chem., 53 (1981) 778-782. 29 G. C. Chen and J. T. Yang, Two point calibration of circular dichrometer with dol0camphorsulfonic acid, Anal. Lett., 10 (1977) 1195-1207. 30 W. C. Krueger and L. M. Pschigoda, Circular dichrometer calibration by Kramers--KrSnlg transform methods, Anal. Chem,, 43 (1971) 675--677. 31 G. C. Kresheck, A. B. Adade and G. Vanderkooi, Differential scanning calorimetry study of local anesthetic effects of F1ATPase and submitochondrtal particles, Biochvm/st~j, 24 (1985) 1715-1719. 32 S. P. Manly, K. S. Matthews and J. M. Sturtevant, Thermal denaturation of the core protein of lac repressor, Biochemistry, 24 (1985) 3842-3846.
302 33 J. Hermaus, Jr. and H. A. Scheraga, Structural studies of ribonuclease. V. Reversible change of configuration, J. Am~ Chem. 8oc., 83 (1961) 3283--3292. 34 K. Takahashi, J. L. Casey and J. M. Sturtevant, Thermodynamics of the binding of D-glucose to yeast hexokinasc, Biochemistry, 20 (1981) 4693-4697. 35 G. C. Kresheck and J. E. Erman, Calorimetric studies of the thermal denaturation of cytochrome c peroxidase, Biochemistry, 27 (1988) 2490--2496. 36 K. Takahashi and J. M. Sturtevant, Thermal denaturation of streptomyces subtilisin inhibitor, subtilisin-BPN', and the inhibitor subtilisin complex, Bioc~mistry, 26 (1981) 178-182. 37 C. T. Chang, C.-S. C. Wu and J. T. Yang, Circular dichroic analysis of protein conformation: inclusion of the j~-turns, Aria/. Biochem., 91 (1978) 13-31. 38 N. J. Gibson and J. Y. Cassim, Evidence for an a.~-type helical conformation for bacteriorhodopsin in the purple membrane, Biochemistry, 28 (1989) 2134-2139. 39 Y. H. Chen, J. T. Yang and K. H. Chau, Determination of the helix and ~ form of proteins in aqueous solution by circular dichroism, Biochemistry, 13 (1974) 3350--3359. 40 D. W. Urry, Protein conformation in biomembranes: optical rotation and absorption of membrane suspensions, Biochim. Biophys. Acta, 265 (1972) 115--168. 41 B. M. Becher and J. Y. Cassim, Effects of light adaptation on the purple membrane structure of Halobacterium holobium, Biophys. J., 16 (1976) 1183-1200. 42 K. Hiraki, T. Hamanaka, T. Mitsul and Y. Kito, Phase transitions of the purple membrane and the brown holo-membrane: X-ray diffraction, circular dichroism spectrum and absorption spectrum studies, Biochim. Biophys. Acta~ 647 (1981) 18-28. 43 B. Zubov, K. Ts~i and B. Hess, Transition kinetics of the conversion of blue to purple bacteriorhodopsin upon magnesium binding, FEBS Lett., 200 (1986) 226--230. 44 M. Ariki and J. K. Lanyi, Characterization of metal ion-bonding sites in bacteriorhodopsin, J. BioL Chem., 261 (1986) 8167-8174.
289
THE THERMAL STABILITY OF NATIVE, DELIPIDATED, DEIONIZED AND REGENERATED BACTERIORHODOPSIN G. C. KRESHECK,C. T. LIN*, L. N. WILLIAMSONand W. R. MASON Depa~ment of Chemistry, Northern Illinois University, DeIfalb, IL 50115 (U.S.A.)
D.-J. JANG and M. A. EL-SAYED Depa.,~.,~nt of Chemistry and Biochemistry, University of Californio~ Los Angeles, CA 90024 (U.S.A.)
(Received October 16, 1989; accepted January 25, 1990)
K e y w o r d s . Thermal stability, Bacteriorhodopsin, deprotonation.
Summary Differential scanning calorimetry curves and circular dichroism spectra were determined for native bacteriorhodopsin (bR), deionized bR, acid blue and acid purple bR, 75% delipidated bR, deionized-delipidated bR and Mg~+ regenerated deionized bR. The effects of the different perturbations on the thermal stability (melting temperature) and the apparent helical content of the protein were examined. These perturbations have more influence on the deprotonation efficiency and the color change of retinal than on the thermal stability and the apparent helical content of the protein. Although the addition of Mg 2+ to deionized bR restores the photochemical cycle, it does not restore the thermal stability to that of the native material.
I. Introduction
The retinylidene protein bacteriorhodopsin ('oR), the other photosynthetic system besides chlorophyll, is one of the protein pigments found in the purple membrane of I - I a I o b c w t e r i u m h a I o b i u m [1, 2]. Light-adapted bR contains an all-trans retinal which is covalently bound to the protein via a protonated Schiff base (PSB) [1, 3]. On absorption of visible light, bR undergoes the photochemical cycle [4] h~,
bR56s ps ~ Ke10 ~
L550
--H + 60 ~ ' M412
+H + ms ' Oe40
'
bR56s
AS a result, the PSB is deprotonated during the L550-* M412 trzlisition, leading to a proton pumping process that increases the proton concentration on the ~Author to whom correspondence should be addressed. Elsevier Sequoia/Printed in The Netherlands
290 outside surface of the membrane [5]. The proton gradient created across the membrane is then used to transform ADP into ATP in the final step of photosynthesis [6]. Understanding the molecular mechanism of deprotonation of the PSB thus becomes very important to our understanding of the molecular mechanism of solar energy storage in nature. Recently, the effects of various perturbations on the probability of the deprotonation process have been studied in detail by a number of research groups. Acidification (to a pH below 3) or removal of cations from bR produces a blue form o f b R [7-10]. This form is incapable of deprotonating to form M41e [11], but still allows the isomerization of retinal and the formation of the IQ]o and L55o analogues [11, 12] to take place. The purple color of bR returns on further acidification of acid blue bR; however, although acid purple bR has K610 and L550 analogues, it does not form M41e nor does it pump protons [11]. Changing the concentration of metal cations in bR is found to change the deprotonation probability non-linearly, but does not change the relative population of metal cation (i.e. Eu 3+) sites [13]. This suggests an indirect involvement of metal cations in the deprotonation process. Corcoran et al. [ 13 ] have observed that most of the luminescence of the bound Eu 8+ originates from surface sites. This observation suggests an indirect involvement of surface metal cations in the control of the protein conformational changes, which have previously been proposed [14, 15] as a mechanism for controlling the rate of the deprotonation process. Following retinal absorption [16, 17] and deprotonation probability of the PSB [18], the effect of removing 7596 of the bR lipids (to form delipidated bR) has been studied as a function of pH and metal cation concentration. The results of these studies provide further indirect evidence of the importance of surface charges in controlling the protein conformation (and thus the retinal color) [16, 17] and protein conformational changes during the photochemical cycle (and thus the deprotonation rate) [14, 15, 18]. The above discussion strongly indicates that protein structure plays a dominant role in the mechanism of the deprotonation process. It has previously been shown [ 19-21 ] that a thermodynamic analysis of the structural stability of bR can be obtained from studies of the thermal unfolding by differential scanning calorimetry (DSC). Previous circular dichroism (CD) studies of bR in various regions of the spectrum have also been used to monitor changes in the environment of the retinal chromophore (visible), the aromatic amino acids (near UV) and the secondary structure of the protein (far UV). These studies have indicated that there are no changes in secondary structure between pH 2.4 and pH 11.8 [22], and that the far-UV CD spectra of lightand dark-adapted bR in solution are identical [23] as are the near-UV CD spectra in acrylamide gels at pH 4 and 7 [8]. The far-UV CD spectra of native and dellpidated oriented films of purple membrane at 15°/0--20% relative humidity have recently been reported to be almost superimposable [24]. In this work, the effect of several different perturbations on the overall macroscopic protein is studied by DSC and CD spectrometry. A comparison is
291 then made between the response to these different perturbations of the protein stability and helical content on the one hand and the structure and function of the active site (as measured by the changes in the deprotonation efficiency) on the other.
2. Experimental details The procedure used for the growth and purification of the purple membrane was a combination of the methods outlined by Oesterhelt and Stoeckenius [25] and Becher and Cassim [26]. The deionized bR samples were prepared by flowing bE through a cation exchange column and washing several times with double deionized water. The delipidated bR was prepared by incubating 10 mg of purple membrane sample overnight at room temperature in 5 ml of 20 mM 3-[(3-cholaxnidopropyl)dimethylanu~onio]-l-propanesulfonate (CHAPS; Pierce Chemical Co., Rockford, IL) containing 5 m_M acetate buffer CpH 5.4) [16]. Deionized distilled water was used to prepare all solutions, and samples were used promptly or stored in polyethylene bottles at 4 °C. Unbuffered bR suspensions which were not deionized gave a measured pH of 5 . 5 ± 0 . 5 , whereas the pH of the deionized samples was slightly lower, e.g. 4 . 0 ± 0.5. These solutions were used for DSC and CD studies without additional buffering components in order to provide the same conditions as used in previous photochemical investigations [18]. Protein concentration was determined from the optical absorption at 280 nm using an extinction coefficient of 113 400 M -1 cm -1. The concentration of delipidated samples was corrected for the small absorbance change (7%) that accompanies removal of phospholipid from purple membrane [24]. The absorbance at 222 nm was also used to corroborate the concentration for all protein solutions. The CD spectra were determined in ambient conditions, 22 ± 1 °C, using a computer-controlled spectrometer built at Northern Illinois University and described elsewhere [27]. The path length of the quartz cells used for the CD measurements was 1.00 cm and the CD scale was calibrated using camphor sulfuric acid solutions [28--30]. The molar ellipticity was calculated using a mean residue weight of 105. The observed differential absorbance ranged from approximately 5 × 1 0 -5 to 1 . 6 × 1 0 -a with an average noise level of 5 × 10 -~ from 190 to 430 nm. For the determination of the melting point by observing color changes from purple (or blue) to yellow, the pH was maintained between pH 3 and 7 with 0.1 M acetate buffer, but was adjusted with HC1 below pH 3. All solutions of native bR for which the pH was 2.5 or below exhibited visual evidence of aggregation. A spectrophotometric method was also used to monitor the effect of temperature on the disappearance of the visible absorption band of bE. The results were the same as those obtained from the visual "melting point" observation. The calorimetric studies were carried out using an MC-1 differential scanning calorimeter (Micro Cal, North Hampton, MA) which was interfaced
292 to an IBM 9000 microcomputer system as previously described [31 ]. Sample and solvent reference solutions of 0.61 ml were added to matched stainless steel cells (Hastelloy C-276) and data were collected at a heating rate of 1.088 K min -1 except for a few runs at a heating rate of 0.357 K min -1. The transition temperature tm (°C) or Tm (K) corresponds to the temperature of maximum excess heat capacity for the transition. No dependence of tm on the scanning rate was noted for any of the samples studied. The total enthalpy change Ah (cal g-1) was found from the area under the peak using an electrical pulse to convert the observed area to units of calories. Numerical integration of the peaks was performed using linear baselines connecting points just below and above the temperature observed for the transitions. The molar calorimetric enthalpy change h/-/¢ was obtained as the product of Ah and the molecular weight of bR (27 000 g mol-1). The van't Hoff enthalpy change AHv~ was estimated assuming a two-state process from the equation [32 ] ~-tvH ----- 4RTm2Cpm/Ah
(1)
where Cp~. is the maximum excess specific heat observed at t~. In most cases, the data above t~ were not symmetrical with respect to the data below tm due to deviations from simple two-state behavior. No attempt was made to perform additional curve fitting due to the unknown state of association of the protein before and after thermal denaturation.
3. R e s u l t s
The temperatures at which the native bR solutions turned yellow in the pH range 0.56-6.50 are given in Fig. 1. This temperature is found to correlate with the transition temperature for the thermal denaturation of the protein, at least in the pH range 5 - 6 used in our DSC studies. The decrease in apparent stability of bR with decreasing pH, as shown by a lower tin, is not unexpected for proteins [33]. The possible loss of metal ions associated with the chromophore and/or the protonation of acidic residues near pH 3.0 are indicated by the blue color of the unheated solutions. The apparent increase in stability below pH 1.5, indicated by an increase in tin, is unexpected. The bR solutions in this pH region are again purple in color at room temperature, but have a CD spectrum very similar to heat-denatured protein. There are at least two possible sources that might account for the increase in the thermal stability of acid purple bR: (i) the protonation of the phospholipid head groups (pKa < 2.5) takes place leading to a change in secondary structure; (ii) the aggregation affects the surface amino acids. The fact that no enhancement in the thermal stability is observed in Fig. 1 for acid blue bR (pH 2.5), where aggregation is also apparent, suggests the importance of secondary structure change in the thermal stability of bR. The alteration of the active site in acid purple bR is such that the isomerization of retinal and formation of Kelo and L55o occur, but deprotonation does not take place.
293
100
90
80
tm(°C) 70
60
50
'
42
2.8
5.6
7.0
pH Fig. 1. Plot of the melting t e m p e r a t u r e tm vs. pH for native bR b a s e d o n the appearance of a yellow solution. The line drawn is empirical.
3.1. D S C s t u d i e s The thermal stabilities of native, delipidated and deionized-delipidated bR were investigated, and endotherms were observed with tm in the range 9 0 - 1 0 0 °C (the b o t t o m three curves of Fig. 2) where the solutions changed color from purple to yellow. All three curves were asymmetrical, with the suggestion of a possible exothermic step above the midpoint t e m p e r a t u r e for the transitions that could result from protein association effects [34]. The thermal denaturation of bR was found to be irreversible from the absence of any transition for samples which were cooled to r o o m t e m p e r a t u r e on completion of the DSC scans and rerun. A summary of the t herm odynam i c data is given in the first four columns of Table 1. The data were analyzed by assuming a single two-step protein denaturation mechanism. By fitting the data up to the t em perat ure of m a x i m u m heat capacity, using the p r o c e d u r e of Kresheck and E rm an [35], the theoretical curves (broken lines) in Fig. 2 were obtained; the differences bet w een the observed and calculated curves were also plotted along the linear baseline drawn between the t e m p e r a t u r e just below and above the transition temperature. The deviations of the residuals from the linear baseline up to tm are comparable with the noise level of the baseline. The sharp decrease in the residuals below the linear baseline above tm reflects the effect of the possible exothermic step indicated above in addition to deviations from the assumed two-step mechanism. The values of AHc and AHvn for representative runs are given in the fifth and sixth columns of Table 1 for the native, delipidated, deiordzed-delipidated, deionized and regenerat ed samples. The van't Hoff enthalpy change is about 33% greater than the calorimetric enthalpy change for the native protein and more than twice as large for the delipidated
294
Delipidated
pH 25
8:1 Mg2+ >}< Q. < O I..< LU "1" (,9 o0 LU O X LU
4:1 Mg2+
Deionized
Deionized- . ~ delipidated
Delipidated
,f~--m~- f~
~,~_
/
~
',
---~-~~'~-'~ j/r~,
Native
2'0
4'0
6'0
8'0
~~0
TEMP (DEG°C) Fig. 2. DSC curves for the thermal denaturation of bR in the native (4.68 g 1-1), delipidated (4.68 g l - l ) , deionized-deUpidated (4.68 g 1-1), deionized (3.14 g l - l ) , regenerated (4:1 Mg~+ (3.14 g 1-1); 8:1 Mg~+ (3.07 g 1-1)) and acid delipidated (pH 2.5) (2.34 g 1-1) states. Each division on the ordinate corresponds to 2.87 mcal K -1 at a scanning rate of 1.088 K rain -~. The calibration pulses around 45 °C for the deionized-delipidated samples correspond to 11.15 mcal. The smooth broken line that follows the sample was calculated according to the procedure described in the text. The linear b r o k e n baselines were used as reference lines for plotting the residuals between the observed and theoretical curves.
samples. The presence of intermolecular steps in the denaturation process is indicated when AHvH exceeds AH c [36]. It is not within the scope of this study to address this question in more detail due to the unknown state of association of the samples, which are known from the literature to be suspensions rather than true solutions. The thermal stabilities of deionized samples of bR was investigated, and results for a representative sample are given in Fig. 2 (fifth curve from the top). Three major differences between the deionized and native samples may
295
c~ i
0
0
0 0 cO
O0 00 ~Ob-
cO
I
2g 00
I
•~
I
I
t
I
1
0
0 c~
0
0
~
-e i
0
I
c~ O o
c~
Lo
¢o
~oo~
c~ e~
0
A
°
~
Oc~oO
P, 0
/
296 be noted. The peak for the deionized sample is smaller, m ore symmetrical and occurs at a lower t em pe r at ur e than the peak for the native sample. In addition, attempts were made to regenerate the native form of bR starting with the deionized state by restoring Mg 2÷ ions. The DSC spect ra obtained are given in Fig. 2 (second and third curves from the top) for 8:1 and 4:1 Mg 2+ respectively, for samples which were regenerat ed and aged 24 h at 4 °C before analysis. It should be not ed that these curves in the middle of the figure are amplified 2.5 times, i.e. the two deionized curves in the middle of the figure are the same e x c e p t for the scale difference. The addition of 4:1 Mg 2÷ restores the original purple color and photocycle of bR but only increases tm slightly. The values of AHc and AHv~ obtained for these samples are also given in the fifth and sixth columns of Table 1. The curve at the top of Fig. 2 represents the scan obtained for a delipidated sample that was adjusted to pH 2.5 (scale amplified 2 times). This sample was purple in color and showed no signs of phase separation, in contrast with the native protein which turns blue and precipitates. The delipidated sample also gave data with a good signal-to-noise ratio, and exhibited a small narrow peak around 90 °C. Under the same conditions, native bR showed a very ragged trace with no sign of a thermal transition within our experimental sensitivity setting. 3.2. CD s p ectr a The CD spectra of native, thermal denatured, acid blue (pH 2.5), acid purple (pH 0), deionized and regenerated samples were determined in order to obtain an indication of the changes in the secondary structure for the different samples. The results from these studies are given in the Iast column of Table 1, together with reference data for [0]222 [37] for the a helix, fl form, fl turns and u n o r d e r e d state as they occur in typical globular proteins. for bR, there is compelling evidence [38] that some of the a helices may be in the an form, rather than exclusively in the a~ form. The an form, due to the tilting of its amide plane relative to the helical axis, exhibits much less ellipticity for an identical helical content than the a~ form in which the amide plane is parallel to the helical axis. As expected, the value of [01222 for the native form of bR indicates the pr esence of a considerable a-helical content. No significant difference in s e c onda ry structure is suggested for the native, deionized, delipidated and r egener a t ed samples. Both the heat- and acid-treated samples appear to have lost a portion of their a-helical content, and the values of [0122z are less than the value of - 13 800 e x p e c t e d for an unor de r ed sample. It is important to note that [01222 for the u n o r d e r e d form in proteins varies over a very wide range of values. The value - 13 800 referred to in Table 1 was obtained by averaging the values for 15 extrinsic proteins. When the values for only five of the 15 extrinsic proteins were used, an average value of about + 1580 was obtained [39]. The effects of aggregation on the CD measurements of bR at pH 0 and 2.5 are uncertain. Figure 3 shows the CD (top) and absorption (bottom)
297 1.0
10 s/kA @.@
I
2@0.@
I
[email protected]
240.@
(rim)
ABS
I.8 1 ~ s B .5
@.0
.
~ i 2B@. I~
i 22B. B
i 24@. @
~k.Cnm) Fig. 3. CD (top) and absorption (bottom) spectra of 5.2x10 -7 M bR in water: (1) native purple bR; (2) heat-treated (or denatured) yellow bR; (3) acid blue bR (pH 2.5); (4) acid purple bR (pH 0); (5) deionized blue bR. s p e c t r a of 5 . 2 x 1 0 - ? M bR in water: (1) native, (2) t h e r m a l l y d e n a t u r e d , (3) acid blue (pH 2.5), (4) acid p u r p l e (pH 0) a n d (5) deionized. Several o b s e r v a t i o n s can b e s u m m a r i z e d . (i) As m e n t i o n e d in S e c t i o n 2, all solutions for w h i c h the p H is 2.5 a n d b e l o w exhibit visual e v i d e n c e o f aggregation. H o w e v e r , at this low c o n c e n t r a t i o n o f 5 . 2 x 1 0 -7 M, all five s a m p l e s s e e m to s h o w a h o m o g e n e o u s solution with n o visible p a r t i c u l a t e s u s p e n s i o n . (ii) The a b s o r b a n c e at ; t - - 2 2 2 n m for all five s a m p l e s is r o u g h l y the same, i . e . A -- 0.5. At A----222 nm, the AA value for e a c h o f the five s a m p l e s (top p o r t i o n o f Fig. 3) is quite distinct. T h e v a l u e s o f [e]222 are listed in Table 1.
298 (iii) In the b o t t o m spectra of Fig. 3, curves 3 and 4 for bR at acidic pH show greater absorbance in the region above 230 nm and lower absorbance around 200 nm. The former may reflect the greater scattering as a result of aggregation and the latter may be due to the Duysens absorption flattening effect which is a concentration obscuring phenomenon. (iv) In the top spectra of Fig. 3, a 70% reduction in ellipticity at maximum absorbance wavelength (A = 200 nm) is observed for acid blue and acid purple hR. Curves 3 and 4 show a depression of the 208 nm CD band and a change in cross-over wavelengths to longer wavelengths. The effect is greater at pH 0 than at pH 2.5 due to differential Mie light scattering. (v) Curve 2 in Fig. 3 shows an approximate 109/0 increase in absorption and 30% decrease in ellipticity at the extreme, indicating some conformational changes for the heat-treated samples. Observations (iii) and (iv) are quite similar to the changes observed in the absorption and CD spectra due to a pH change from 3.85 to 2.4 in helical polyglutamic acid suspensions [40]. Urry [40] has described a pr oc e dur e for estimating the molar ellipticity at 222 nm of polypeptides that exist as suspensions rather than molecularly dispersed solutions. He notes the following at 222 urn: (a) the effects due to differential scattering of left and right circularly polarized light are zero; (b) the absorbance of a sample adequate to penetrate the UV to 192 um would be low, such that the Duysens absorption flattening coefficient would be close to unity; (c) the absorbance at 222 nm exhibits little h y p e r c h r o m i s m or hypochromism on changing protein conformation. In the light of these facts, observation (ii) and the results in Fig. 1, it is suggested that the aggregation is not the only factor responsible for the [0]222 value observed in acid blue and acid purple bR.
4. D i s c u s s i o n a n d c o n c l u s i o n s The [ 0]222 value obtained for native bR ( - 15 600 :i=600 deg cm 2 d m o l - 1) compares well with the value of - 15 900 deg cm 2 d m o l - 1 at 223 nm r e p o r t e d by Becher and Cassim [41]. In addition, our observation that delipidation does not significantly alter the value of [0]222 is in accord with the findings of Hartsel and Cassim [24], although Triton X-100 was used for lipid removal in their work and CHAPS in ours. However, we do not concur with the hterature with r es pe c t to the effects of acidification to pH 2 . 4 - 2 . 5 on [ 0]222. Muccio and Cassim [22] r e por t ed that this value is unchanged between pH 2.4 and 5.0, whereas our value decreased from - 1 5 600 to - 9 1 0 0 deg cm 2 dmo1-1 with a decrease in pH from 5.5 :fi0.5 to 2.5. In spite of the aggregation observed in the acid blue and acid purple bR samples, it is difficult to explain this discrepancy since Muccio and Cassim not ed that optical artifacts due to absorption flattening, light scattering and the differential hght-scattering effect of circularly polarized light are minimal at 222 rim.
299 However, our results at 222 nm (which reflect the contribution of the peptide backbone) parallel the results of Mowery et al. [8] at 315 a m (which reflects the contribution of the retinal chromophore), where the pH 2.0 spectrum showed a marked decrease in ellipticity at 315 nm when compared with the ellipticity at pH 4 or 7. Our results thus suggest a possible protein conformational contribution to other known changes in physical and chemical behavior of bR between pH 0 and pH 2.5 [8]. The thermodynamic data (tin, AHc and AHvu) obtained in this study for native bR in water are in good agreement with the results obtained by previous workers [19-21]. Our value of tm (97.1 ± 1.4 °C) for the "100 °C" transition is closest to that of Jackson and Sturtevant [19] ( 9 5 . 0 ± 0 . 7 °C), but our AH¢ value (92 ± 3 kcal mol-1) is closest to that reported by TristramNagle et al. [20] (91___9 kcal mol-~). It is noted that the AH¢ value given is an average of five runs, whereas those reported in Table 1 are representative values calculated from a single scan. Jackson and Sturtevant [ 19 ] found that the AHvH/AH¢ ratio was slightly less than 2 regardless of ionic conditions, and Brouillette et al. [21 ] obtained an average value of 1.9 from data collected between pH 6 and pH 11. The value we obtained for native bR in this study was also 1.9, again suggesting possible intermolecular cooperativity in the thermal denaturation of bR. The so-called "80 °C" pretransition was only evident in our most concentrated sample (5.8 mg m1-1) and it was located at 78.4 °C with a calorimetric enthalpy change of 9.7 kcal mol-x. This compares well with a temperature of 79.4 ± 0.8 °C and 6.50 ± 0.63 kcal m o l - 1 in water reported by Jackson and Sturtevant [19] for tm and AH¢ respectively for this transition. It is of interest that a purple to reddish-purple color change was observed visually at about 70 °C, which corresponds to the onset of this "pretransition". The significance of this color change is not known, but it could be due to the loss of crystallim'ty of the bR lattice which occurs at about 80 °C as reported by Hiraki et al. [42]. The effect of increasing the hydrogen ion concentration is first to decrease the melting point from 97 °C in bR until acid blue bR is formed (melting point = 65 °C). A further decrease in pH leads to an increase in the melting point as acid purple bR is formed (melting point above 90 °C; similar to native bR). Although acid purple bR resembles native bR in having a similar absorption maximum, similar melting point and similar initial steps in the photocycle (isomerization of retinal [ 12] and the formation of K82o and L55o [ 11 ]), acid purple bR has far less ~-helix content than native bR. Furthermore, the PSB in acid purple bR does not deprotonate during the cycle. This suggests that the deprotonation process, unlike the isomerization or melting temperature, is more sensitive to the conformational changes of the protein than the formation of the K~lo or Lsso forms during the cycle. This is consistent with the previous conclusion [ 13, 18 ] that the protonation process is controlled by the nature of the protein conformational change during the L550-~M4~ process (in contrast with the intra-retinal isomerization which occurs during the b R - ) K61o~ Lsso processes).
300 T h e a b o v e o b s e r v a t i o n s s u g g e s t t h a t the t h e r m a l stability of the p r o t e i n d o e s n o t c o r r e l a t e with t h e d e p r o t o n a t i o n ability o f the PSB. T h e d e n a t u r a t i o n t r a n s i t i o n t e m p e r a t u r e s o f native bR a n d d e l i p i d a t e d bR a r e f o u n d to b e similar, b u t t h e i r d e p r o t o n a t i o n efficiencies differ b y 50%. As e x p e c t e d , delipidation d o e s n o t c h a n g e the t h e r m a l stability or the a-helix c o n t e n t ( o r the s e c o n d a r y s t r u c t u r e ) o f t h e p r o t e i n at p H 5 . 5 ± 0 . 5 . H o w e v e r , it d o e s c h a n g e t h e solubility at p H 2.5 a n d t h e c h a r g e distribution [16, 17] w h i c h c a n h a v e effects on c o n f o r m a t i o n a l c h a n g e s d u r i n g the p h o t o c h e m i c a l cycle [18]. D e i o n i z a t i o n is f o u n d to d e c r e a s e the t h e r m a l stability o f t h e p r o t e i n as s h o w n in T a b l e 1. T h e t r a n s i t i o n t e m p e r a t u r e is a p p r o x i m a t e l y 70 °C, n o t far f r o m the acid b l u e bR m e l t i n g p o i n t ( a p p r o x i m a t e l y 65 °C) m e a s u r e d b y the visual t e c h n i q u e (Fig. 1). T h e e n t h a l p y o f d e n a t u r a t i o n o f deionized o r acid b l u e bR is o v e r 15% less t h a n t h a t for native bR. B o t h acid b l u e a n d deionized bR h a v e retinals t h a t i s o m e r i z e , b u t t h e i r PSB d o e s n o t d e p r o t o n a t e . T h e r e c o v e r y o f the d e p r o t o n a t i o n efficiency o f t h e PSB a n d t h e p u r p l e c o l o r o f bR o n addition o f c a t i o n s to d e i o n i z e d bR is f o u n d to b e r a p i d ( m i l l i s e c o n d s ) [43] a n d n o n - s p e c i f i c [10, 44].
Acknowledgments G. C. K. t h a n k s the NSF f o r s u p p o r t ( G r a n t No. P R M - 8 1 1 3 3 7 7 ) . C. T. L. w i s h e s to a c k n o w l e d g e the financial s u p p o r t f r o m t h e N o r t h e r n Illinois University G r a d u a t e S c h o o l a n d College o f Liberal Arts a n d Sciences. M. A. E. a n d D. J. J. wish to t h a n k the D e p a r t m e n t o f E n e r g y (Office o f B a s i c E n e r g y Sciences; G r a n t No. D E - F G 0 3 - 8 8 E R 1 3 8 2 8 ) f o r financial s u p p o r t .
References 1 D. Oesterhelt and W. Stoeckenius, Rhodopsin-like protein from the purple membrane of Halobacterium halobium, Nature ~ ) , New BIOL, 233 (1971) 149-152. 2 W. Stoeckenius and R. A. Bogomolni, Bacteriorhodopsin and related pigments of halobaeteria, Annu~ Rev. Biochem., 52 (1982) 587--619. 3 J. Bridgen and I. D. Walker, Photoreceptor protein from the purple membrane of Halobacterium hah~bium: molecular weight and retinal binding site, Biochemistry, 15 (1976) 792-798. 4 R. Lozier, R. A. Bogomolni and W. Stoeckenius, Bacteriorhodopsin: a light driven proton pump in Halobacterium halobium, Biophys. J., 15 (1975) 955--962. 5 N. Dencher and M. W'flms, Flash photometric experiments on the photochemical cycle of bacteriorhodopsin, Biophys. Struct. Mech., 1 (1975) 259-271. 6 J. W. Belliveau and J. K. Lanyi, Analogies between respiration and a light-driven proton pump as sources of energy for active glutamate transport in Halobacterium holobiur~ Arch. Biochem~ Biophys., 178 (1977) 308-314. 7 T. A. Moore, M. E. Edgerton, G. Parr, C. Greenwood and R. N. Perham, Studies of an acid-induced species of purple membrane from Halobacterium halobium, Biochem. J., 171 (1978) 469--476. 8 P. C. Mowery, R. H. Lozier, Q. Chae, Y.-W. Tseng, M. Taylor and W. Stoeckenins, Effect of acid pH on the absorption spectra and photoreactions of bacteriorhodopsin, Biochemistry, 18 (1979) 4100---4107.
301 9 Y. Kimura, A. Ikegami and V~. Stoeckenius, Salt and pH dependent changes of the purple membrane absorption spectrum, Photochern. Photobiol., 40 (1984) 641-646. 10 C. H. Chang, J.-G. Chen, R. Govindjee and T. Ebery, Cation binding by bacteriorhodopsin, Proc. Natl. Acad. Sci. USA, 82 (1985) 396-400. 11 E. L. Chronister, T. C. Corcoran, L. Song and M. A. E1-Sayed, On the molecular mechanisms of the Sehiff base deprotonation during the bacteriorhodopsin photocycle, Proc. Natl. Acad. Sci. USA, 83 (1986) 8580--8584. 12 T. Kobayashi, H. Ohtani, J. Iwai, A. Ikegami and H. Uehiki, Effect ofpH on the photoreaction cycles of bacteriorhodopsin, FEBS Lett., 162 (1983) 197-200. 13 T. C. Corcoran, K. Z. Ismail and M. A. E1-Sayed, Evidence for the involvement of more than one metal cation in the Schiff base deprotonation process during the photocycle of bacteriorhodopsin, Proc. Natl. Acad~ Sci. USA, 84 (1987) 4094-4098. 14 P. Dupuis, T. C. Corcoran and M. A. El-Sayed, Importance of bound cations to the tyrosine deprotonation during the photocycle of bacteriorhodopsin, Proc. Nat/. Acad. Sc/. USA, 82 (1985) 3662-3664. 15 J. H. Hanamoto, P. Dupuis and M. A. EI-Sayed, On the protein (tyrosine)--chromophore (protonated Schiff base) coupling in bacteriorhodopsin, Proc. Natl. Acad. Sci. US~ 81 (1984) 7083--7087. 16 I. Szundi and W. Stoeckenins, Effect of lipid surface changes on purple to blue transition of bacteriorhodopsin, Proc. Natl. Acad. Sci. USA, 84 (1987) 3681-3684. 17 I. Szundi and W. Stoeckenius, Purple-to-blue transition of bacteriorhodopsin in a neutral lipid environment, Biophys. J., 54 (1988) 227-232. 18 D. J. Jang and M. A. El-Sayed, Deprotonation of lipid-depleted bacteriorhodopsin, Proc. Natl. Acad. Sci. US~ 85 (1988) 5918--5922. 19 M. B. Jackson and J. N. Sturtevant, Phase transition of the purple membrane of Ha/obacterium, Biochemistry, 17 (1978) 911-915. 20 S. Tristram-Nagle, C. P. Yang and J. F. Nagle, Thermodynamic studies of purple membrane, Biochim. Biophys. Acta, 854 (1986) 58-66. 21 C. G. Brouillette, D. D. Muccio and T. K. Finney, pH dependence of bacteriorhodopsin thermal unfolding, Biochemistry, 26 (1987) 7431-7438. 22 D. D. Muccio and J. Y. Cassim, Interpretations of the effects of pH on the spectra of purple membrane, J. MoL Biol., 135 (1979) 595-609. 23 J. E. Draheim and J. Y. Cassim, Large scale global structural changes of the purple membrane during the photocycle, Biophys. J., 47 (1985) 497-507. 24 S. C. Hartsel and J. Y. Cassim, Structure and photodynamics of bacteriorhodopstn in a delipidated contracted lattice form of purple membrane, Biochemistry, 27 (1988) 3720-3724. 25 D. Oesterhelt and W. Stoeckenius, Isolation of the cell membrane of Halobacterium halobium and its fractionation into red and purple membrane, Methods Ene.~jmol., 31 (1974) 667-678. 26 B. M. Becher and J. Y. Cassim, Improved isolation procedures for the purple membrane of Halobacterium halobium, Prep. Biochern., 5 (1975) 161-178. 27 W. R. Mason, Spectrometer for simultaneous measurement of absorption and circular dichroism spectra, A n a l Chem., 54 (1982) 646-648. 28 P. H. Schippers and H. P. J. M. Dekkers, Direct determination of absolute circular dichroism data and calibration of commercial instruments, Anal. Chem., 53 (1981) 778-782. 29 G. C. Chen and J. T. Yang, Two point calibration of circular dichrometer with dol0camphorsulfonic acid, Anal. Lett., 10 (1977) 1195-1207. 30 W. C. Krueger and L. M. Pschigoda, Circular dichrometer calibration by Kramers--KrSnlg transform methods, Anal. Chem,, 43 (1971) 675--677. 31 G. C. Kresheck, A. B. Adade and G. Vanderkooi, Differential scanning calorimetry study of local anesthetic effects of F1ATPase and submitochondrtal particles, Biochvm/st~j, 24 (1985) 1715-1719. 32 S. P. Manly, K. S. Matthews and J. M. Sturtevant, Thermal denaturation of the core protein of lac repressor, Biochemistry, 24 (1985) 3842-3846.
302 33 J. Hermaus, Jr. and H. A. Scheraga, Structural studies of ribonuclease. V. Reversible change of configuration, J. Am~ Chem. 8oc., 83 (1961) 3283--3292. 34 K. Takahashi, J. L. Casey and J. M. Sturtevant, Thermodynamics of the binding of D-glucose to yeast hexokinasc, Biochemistry, 20 (1981) 4693-4697. 35 G. C. Kresheck and J. E. Erman, Calorimetric studies of the thermal denaturation of cytochrome c peroxidase, Biochemistry, 27 (1988) 2490--2496. 36 K. Takahashi and J. M. Sturtevant, Thermal denaturation of streptomyces subtilisin inhibitor, subtilisin-BPN', and the inhibitor subtilisin complex, Bioc~mistry, 26 (1981) 178-182. 37 C. T. Chang, C.-S. C. Wu and J. T. Yang, Circular dichroic analysis of protein conformation: inclusion of the j~-turns, Aria/. Biochem., 91 (1978) 13-31. 38 N. J. Gibson and J. Y. Cassim, Evidence for an a.~-type helical conformation for bacteriorhodopsin in the purple membrane, Biochemistry, 28 (1989) 2134-2139. 39 Y. H. Chen, J. T. Yang and K. H. Chau, Determination of the helix and ~ form of proteins in aqueous solution by circular dichroism, Biochemistry, 13 (1974) 3350--3359. 40 D. W. Urry, Protein conformation in biomembranes: optical rotation and absorption of membrane suspensions, Biochim. Biophys. Acta, 265 (1972) 115--168. 41 B. M. Becher and J. Y. Cassim, Effects of light adaptation on the purple membrane structure of Halobacterium holobium, Biophys. J., 16 (1976) 1183-1200. 42 K. Hiraki, T. Hamanaka, T. Mitsul and Y. Kito, Phase transitions of the purple membrane and the brown holo-membrane: X-ray diffraction, circular dichroism spectrum and absorption spectrum studies, Biochim. Biophys. Acta~ 647 (1981) 18-28. 43 B. Zubov, K. Ts~i and B. Hess, Transition kinetics of the conversion of blue to purple bacteriorhodopsin upon magnesium binding, FEBS Lett., 200 (1986) 226--230. 44 M. Ariki and J. K. Lanyi, Characterization of metal ion-bonding sites in bacteriorhodopsin, J. BioL Chem., 261 (1986) 8167-8174.