J. Mol. Biol. (1976) 100, 459-472
Conformational Transitions of the lac Repressor from Escherichia coli FELICIA Y . - H . W u , PRADIP BANDYOPADHYAY AND CHENG-WEN W u
Department of Biophysics, Division of Biological Sciences Albert Einstein College of Medicine Bronx, N . Y . 10461, U.S.A. (Received 10 June 1975) T e m p e r a t u r e - j u m p studies of the lac represser were performed monitoring the intrinsic t r y p t o p h a n fluorescence of the protein. A single relaxation process with a time constant of 1.36 • 104 s - 1 was observed for lac represser solutions at p H 7.5 in the absence of inducer. The relaxation time is independent of the concentration of lac represser indicating a conformational transition between two states of the represser (R* r R). Although the binding of an inducer, isopropyl-fl-D-thiogalactoside, is too slow (as shown b y stopped-flow studies) to influence the rate of the rapid interconversion between these two states of the lac represser, the relaxation amplitude decreases with increasing I P T G ~ concentration. The rate of this conformational transition is altered b y protonation of the represser. T e m p e r a t u r e - j u m p d a t a obtained for lac represser at various p H values can be analyzed in terms of a concerted mechanism in which a proton binds preferentially to one of the two states of lac represser (R). We propose t h a t I P T G also binds selectively to the R state. This is consistent with our observation t h a t addition of 1O-3 M-IPTG shifts the p K of a specific group of amino acid residues in the lac represser. Furthermore, the mechanism provides satisfactory explanations for previous reports t h a t the bimolecular rate-constant for the IPTG-lac represser interaction is extremely low a n d t h a t the inducer binding m a y exhibit various degrees of co-operativity a t different p H values. Thus the allosteric transition of lac represser effected by proton and inducer m a y p l a y a significant role in the regulation of lac transcription. 1. I n t r o d u c t i o n T h e c o n t r o l s y s t e m o f t h e / a c o p e r o n in Escherichia coli has b e e n t h e focus for t h e s t u d y o f gene r e g u l a t i o n in t h e p a s t decade. T h e n e g a t i v e c o n t r o l e l e m e n t o f t h i s s y s t e m , t h e / a c represser, is now a v a i l a b l e in r e a s o n a b l e q u a n t i t i e s (Yliiller-Hill et al., 1968) for p h y s i c o - c h e m i c a l studies. T h e r e p r e s s e r is a t e t r a m e r i c p r o t e i n w i t h molec u l a r w e i g h t of 150,000 (Miiller-Hill et al., 1971 ; B e y r e u t h e r et al., 1973). R e c e n t l y , L a i k e n et al. (1972) h a v e e x p l o i t e d t h e change in t h e t r y p t o p h a n fluorescence o f / a c r e p r e s s e r in e q u i l i b r i u m a n d k i n e t i c studies o f t h e r e p r e s s e r - i n d u c e r i n t e r a c t i o n . T h e s t o p p e d - f l o w k i n e t i c d a t a a r e c o n s i s t e n t w i t h a simple t w o - s t e p m e c h a n i s m in which a b i m o l e e u l a r b i n d i n g o f t h e i n d u c e r (I) t o t h e r e p r e s s e r (R) is followed b y a slow i s o m e r i z a t i o n o f t h e i n d u c e r - r e p r e s s e r c o m p l e x ( R I a n d R I ' ) . R ~- 1
" RI.
" RI'.
(1)
H o w e v e r , t w o of t h e p h y s i c a l consequences o f t h e i r m e c h a n i s m are n o t a p p e a l i n g . First, the bimolecular rate constant (6• M -1 s -1) is t h r e e to four orders o f m a g n i t u d e s m a l l e r t h a n e x p e c t e d for a diffusion-controlled r e a c t i o n i n v o l v i n g t Abbreviation used: IPTG, isopropyl-fl-D-thiogalactoside. 31 459
460
F.Y.-H.
WU, P. B A N D Y O P A D H Y A Y AND C.-W. WU
molecules of the size of t h e / a c represser and the inducer. Second, their mechanism invokes direct contact between the inducer and the t r y p t o p h a n residue of the represser, yet observations with model compound suggest t h a t this will lead to quite a different effect on t r y p t o p h a n fluorescence t h a n t h a t actually observed. The authors have pointed out t h a t both these problems m a y be overcome b y an additional, non-rate-limiting, isomerization o f / a c represser before inducer binding. I R*
fast
' g
gI
" gl'.
(2)
I n this mechanism, the fluorescence change which appears to be associated with the bimolecular step is due to a rapid intereonversion between two existing forms of represser, thus obviating the necessity for direct contact between iuducer and tryptophan. Moreover, if the ratio of equilibrium concentrations of R* and R is of the order of 102, the bimolecular rate constant will approach the usual diffusioncontrolled values. Although a rapid conformational transition of t h e / a c represser as described above is critical in revealing the molecular mechanism, the authors claim t h a t a n y fast process occurring outside the time resolution of the stopped-flow instrument (~-~1 ms) is unlikely to result in significant change of fluorescence, since the entire fluorescence change observed b y equilibrium methods is accounted for b y the kinetic signal observed. I t is often true t h a t the information obtained from equilibrium studies cannot be used to resolve a detailed kinetic mechanism. To further elucidate the molecular mechanism of the represser-inducer interaction, in particular to detect elementary steps in the time range faster t h a n a millisecond, we have undertaken temperaturejump studies o f / a c represser monitoring the intrinsic t r y p t o p h a n fluorescence. We report here t h a t a rapid equilibrium exists between two conformational states of lac represser. Evidence will be presented t h a t this conformational equilibrium is affected by proton and the inducer. The possible role of this t y p e of allosterie transition in gene regulation will be discussed.
2. Materials and Methods (a) Reagents Isopropyl-fl-I)-thiogalactoside was obtained from Calbiocliem and 14C-labeled IPTG~ (17 Ci/mol) was purchased from Schwartz/Mann Biochemical. DNase I (pancreatic) was the electrophoretic grade product from Worthington Biochemical Co. All other chemicals were reagent grade. (b) Buffers All buffers used in purification of lac represser were the same as described by Platt et al. (1973). IPTG-binding assay (giggs & Bourgeois, 1968) was carried out in 0-01 ~t-Tris (pH 7-4), 0.2 ~-KC1, 0.01 M-magnesium acetate, 0.1 ml~-EDTA and 1 mI~-mercaptoethanol. All temperature-jump experiments were performed in buffer I (0.1 M-potassium phosphate, 0.1 m~t-dithiotkreitol and 0.1 ml~-MgC12); p H was adjusted to the desired value in each experiment. (c) Purification of lae represser The represser was isotatod from E. cell K I 2 (strain M96) following the procedure of Platt et al. (1973) with slight modification. The potassium phosphate gradient from 0.10 M to 0.24 M was used in the last phosphoeellulose column. The purified represser See footnote to p. 459.
C O N F O R M A T I O N A L C H A N G E S OF lae R E P R E S S E R
461
revealed a single protein b a n d and was better t h a n 98~o pure as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Represser concentrations were determined using the method of Bticher (1947) and the molar extinction coefficient of 9 x 1 0 4 ~ - 1 cm-1 at 280 n m (Butler et al., 1975). The represser in small portions was stored in 1 M-Tris (pH 7.6), 0.3 mM-dithiothreitol and 30% (v/v) glycerol at --70~ (d) Assay of I P T G binding to the represser I P T G - b i n d i n g activity was determined both b y equilibrium dialysis (Gilbert & MfillerHill, 1966) and b y Millipore filter assay (Riggs & Bourgeois, 1968) using [14C]IPTG (30 ets/rnin per pmol).
(c) Potentiometric titration of lac represser A Radiometer PHM-26 p H meter and a Titrograph SBR2-C were used for the continuous potentiometric titration. The electrodes used were G2222C a n d 4112 (reference). The lac represser was dialyzed extensively against 0"2 M-KC1 solution. I t was i m p o r t a n t to preadjust the p H of the solution slightly above 6 to prevent precipitation of the protein. A portion of the protein solution (2 to 5 mg/ml) was placed in the reaction vessel (0.5 ml). After temperature equilibration (10 ~ or 25~ nitrogen was blown over the surface of the solution during the titration. The titration was performed using 0"05 ~-KOI.I standardized with potassium hydrogen phthalate, the solution being constantly stirred with a magnetic stirrer. As a blank, 0.2 M-KC1 solution was also titrated in the same manner. After correction for the blank, titration results were expressed as moles of proton dissociated per mole of protein (Perlmann, 1972). Discontinuous potentiometric titration was also carried out b y adding portions of K O H to the protein solution. The behavior of the protein was the same under these two different procedures of titration. (f) Spectrophotometric measurements Absorption spectra were measured with a Cary 118-C recording spectrophotometer. Fluorescence spectra were recorded on a n Hitachi P e r k i n - E l m e r MPF-3 spectrofluorometer equipped with a corrected spectra accessory. All spectral measurements were made at 25~ with represser concentrations of at least 10 -6 ~. (g) Temperature-jump experiments The equipment used for the temperature-jump relaxation measurements was a combined stopped-flow temperature-jump apparatus constructed in this laboratory (Wu & Wu, 1974). The reaction cell of this apparatus is about 0-3 ml in volume a n d utilizes two conical lenses a t right-angles to each other. A n exciting wavelength of 295 n m from a 200 W I-Ianovia xenon/mercury arc lamp was isolated with a Bausch a n d Lomb grating monochromator. The emission wavelength was isolated from the exciting wavelength by Corning 7-51 a n d 0-52 filters. The changes in fluorescence wore detected with a n E M I 9635QB photomultiplier tube. Signal-to-noise ratios of 1000~3000 were routinely obtained. Temperature j u m p s of 7.5~ were applied to the solution b y discharge of a 0.1 /~F capacitor which had been charged to 10 kV. The resolution time of the apparatus is approximately 10/~s. The temperature-jump was from 17.5 ~ to 25~ in all eases. The kinetic information was recorded on a Tektronix 543 storage oscilloscope and transferred directly on-line to a PDP-11 digital computer b y a Biomation Model 802 transient recorder. The relaxation times a n d amplitudes were calculated b y a least-squares analysis in terms of one or two relaxation processes. The data of successive measurements on the same reaction mixture were accumulated to enhance the signal-to-noise ratio. I n some eases the oscilloscope traces were photographed a n d analyzed graphically. The results were in good agreement with the computer analysis. The experimental u n c e r t a i n t y in the relaxation times was estimated to be a b o u t 10~/o.
3. Results and Treatment o f Data W h e n t h e fluorescence i n t e n s i t y change a t 360 n m was followed, a single r e l a x a t i o n process could be d e t e c t e d w i t h t h e t e m p e r a t u r e - j u m p for solutions c o n t a i n i n g /ac represser i n buffer I ( p i t 7-5) i n t h e absence of inducer. A t y p i c a l oscilloscope trace
462
F. Y.-H.
WU,
P. BANDYOPADHYAY
AND
C.-W.
WU
of the relaxation process following a temperature-jump is given in Figure 1. No relaxation was seen in the absence of the protein. Figure 2 shows t h a t the reciprocal relaxation time, 1]T ~ 13,600 s -z, associated with this relaxation effect is independent of the concentration o f / a c repressor, suggesting the existence of a conformational equilibrium between two different forms of the repressor. kr
R*.
" R.
(3)
1/T = kf +/c~
(4)
kr
The relaxation time for this mechanism
is not dependent on the repressor concentration. I n the presence of the inducer, isopropyl-fl-D-thiogalactoside, two relaxation effects were seen, a fast one in the time range of about 50 ~s and a slow one in the time range longer t h a n 100 ms. The slower relaxation is probably associated with the bimolecular reaction of I P T G and lac repressor, since it occurs in the time scale similar to t h a t of the bimolecular binding of the same concentrations of these molecules observed b y the stopped-flow measurements (Laiken et al., 1972). However, because of its overlap with the optical changes associated with the convection of the solution, this slow relaxation process cannot be reliably analyzed using temperature-jump procedures. The fast relaxation process is essentially the same as t h a t observed in the absence of the inducer. I n addition, the relaxation time for the fast
tl.
Conformational Transitions of the lac Repressor from Escherichia coli FELICIA Y . - H . W u , PRADIP BANDYOPADHYAY AND CHENG-WEN W u
Department of Biophysics, Division of Biological Sciences Albert Einstein College of Medicine Bronx, N . Y . 10461, U.S.A. (Received 10 June 1975) T e m p e r a t u r e - j u m p studies of the lac represser were performed monitoring the intrinsic t r y p t o p h a n fluorescence of the protein. A single relaxation process with a time constant of 1.36 • 104 s - 1 was observed for lac represser solutions at p H 7.5 in the absence of inducer. The relaxation time is independent of the concentration of lac represser indicating a conformational transition between two states of the represser (R* r R). Although the binding of an inducer, isopropyl-fl-D-thiogalactoside, is too slow (as shown b y stopped-flow studies) to influence the rate of the rapid interconversion between these two states of the lac represser, the relaxation amplitude decreases with increasing I P T G ~ concentration. The rate of this conformational transition is altered b y protonation of the represser. T e m p e r a t u r e - j u m p d a t a obtained for lac represser at various p H values can be analyzed in terms of a concerted mechanism in which a proton binds preferentially to one of the two states of lac represser (R). We propose t h a t I P T G also binds selectively to the R state. This is consistent with our observation t h a t addition of 1O-3 M-IPTG shifts the p K of a specific group of amino acid residues in the lac represser. Furthermore, the mechanism provides satisfactory explanations for previous reports t h a t the bimolecular rate-constant for the IPTG-lac represser interaction is extremely low a n d t h a t the inducer binding m a y exhibit various degrees of co-operativity a t different p H values. Thus the allosteric transition of lac represser effected by proton and inducer m a y p l a y a significant role in the regulation of lac transcription. 1. I n t r o d u c t i o n T h e c o n t r o l s y s t e m o f t h e / a c o p e r o n in Escherichia coli has b e e n t h e focus for t h e s t u d y o f gene r e g u l a t i o n in t h e p a s t decade. T h e n e g a t i v e c o n t r o l e l e m e n t o f t h i s s y s t e m , t h e / a c represser, is now a v a i l a b l e in r e a s o n a b l e q u a n t i t i e s (Yliiller-Hill et al., 1968) for p h y s i c o - c h e m i c a l studies. T h e r e p r e s s e r is a t e t r a m e r i c p r o t e i n w i t h molec u l a r w e i g h t of 150,000 (Miiller-Hill et al., 1971 ; B e y r e u t h e r et al., 1973). R e c e n t l y , L a i k e n et al. (1972) h a v e e x p l o i t e d t h e change in t h e t r y p t o p h a n fluorescence o f / a c r e p r e s s e r in e q u i l i b r i u m a n d k i n e t i c studies o f t h e r e p r e s s e r - i n d u c e r i n t e r a c t i o n . T h e s t o p p e d - f l o w k i n e t i c d a t a a r e c o n s i s t e n t w i t h a simple t w o - s t e p m e c h a n i s m in which a b i m o l e e u l a r b i n d i n g o f t h e i n d u c e r (I) t o t h e r e p r e s s e r (R) is followed b y a slow i s o m e r i z a t i o n o f t h e i n d u c e r - r e p r e s s e r c o m p l e x ( R I a n d R I ' ) . R ~- 1
" RI.
" RI'.
(1)
H o w e v e r , t w o of t h e p h y s i c a l consequences o f t h e i r m e c h a n i s m are n o t a p p e a l i n g . First, the bimolecular rate constant (6• M -1 s -1) is t h r e e to four orders o f m a g n i t u d e s m a l l e r t h a n e x p e c t e d for a diffusion-controlled r e a c t i o n i n v o l v i n g t Abbreviation used: IPTG, isopropyl-fl-D-thiogalactoside. 31 459
460
F.Y.-H.
WU, P. B A N D Y O P A D H Y A Y AND C.-W. WU
molecules of the size of t h e / a c represser and the inducer. Second, their mechanism invokes direct contact between the inducer and the t r y p t o p h a n residue of the represser, yet observations with model compound suggest t h a t this will lead to quite a different effect on t r y p t o p h a n fluorescence t h a n t h a t actually observed. The authors have pointed out t h a t both these problems m a y be overcome b y an additional, non-rate-limiting, isomerization o f / a c represser before inducer binding. I R*
fast
' g
gI
" gl'.
(2)
I n this mechanism, the fluorescence change which appears to be associated with the bimolecular step is due to a rapid intereonversion between two existing forms of represser, thus obviating the necessity for direct contact between iuducer and tryptophan. Moreover, if the ratio of equilibrium concentrations of R* and R is of the order of 102, the bimolecular rate constant will approach the usual diffusioncontrolled values. Although a rapid conformational transition of t h e / a c represser as described above is critical in revealing the molecular mechanism, the authors claim t h a t a n y fast process occurring outside the time resolution of the stopped-flow instrument (~-~1 ms) is unlikely to result in significant change of fluorescence, since the entire fluorescence change observed b y equilibrium methods is accounted for b y the kinetic signal observed. I t is often true t h a t the information obtained from equilibrium studies cannot be used to resolve a detailed kinetic mechanism. To further elucidate the molecular mechanism of the represser-inducer interaction, in particular to detect elementary steps in the time range faster t h a n a millisecond, we have undertaken temperaturejump studies o f / a c represser monitoring the intrinsic t r y p t o p h a n fluorescence. We report here t h a t a rapid equilibrium exists between two conformational states of lac represser. Evidence will be presented t h a t this conformational equilibrium is affected by proton and the inducer. The possible role of this t y p e of allosterie transition in gene regulation will be discussed.
2. Materials and Methods (a) Reagents Isopropyl-fl-I)-thiogalactoside was obtained from Calbiocliem and 14C-labeled IPTG~ (17 Ci/mol) was purchased from Schwartz/Mann Biochemical. DNase I (pancreatic) was the electrophoretic grade product from Worthington Biochemical Co. All other chemicals were reagent grade. (b) Buffers All buffers used in purification of lac represser were the same as described by Platt et al. (1973). IPTG-binding assay (giggs & Bourgeois, 1968) was carried out in 0-01 ~t-Tris (pH 7-4), 0.2 ~-KC1, 0.01 M-magnesium acetate, 0.1 ml~-EDTA and 1 mI~-mercaptoethanol. All temperature-jump experiments were performed in buffer I (0.1 M-potassium phosphate, 0.1 m~t-dithiotkreitol and 0.1 ml~-MgC12); p H was adjusted to the desired value in each experiment. (c) Purification of lae represser The represser was isotatod from E. cell K I 2 (strain M96) following the procedure of Platt et al. (1973) with slight modification. The potassium phosphate gradient from 0.10 M to 0.24 M was used in the last phosphoeellulose column. The purified represser See footnote to p. 459.
C O N F O R M A T I O N A L C H A N G E S OF lae R E P R E S S E R
461
revealed a single protein b a n d and was better t h a n 98~o pure as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Represser concentrations were determined using the method of Bticher (1947) and the molar extinction coefficient of 9 x 1 0 4 ~ - 1 cm-1 at 280 n m (Butler et al., 1975). The represser in small portions was stored in 1 M-Tris (pH 7.6), 0.3 mM-dithiothreitol and 30% (v/v) glycerol at --70~ (d) Assay of I P T G binding to the represser I P T G - b i n d i n g activity was determined both b y equilibrium dialysis (Gilbert & MfillerHill, 1966) and b y Millipore filter assay (Riggs & Bourgeois, 1968) using [14C]IPTG (30 ets/rnin per pmol).
(c) Potentiometric titration of lac represser A Radiometer PHM-26 p H meter and a Titrograph SBR2-C were used for the continuous potentiometric titration. The electrodes used were G2222C a n d 4112 (reference). The lac represser was dialyzed extensively against 0"2 M-KC1 solution. I t was i m p o r t a n t to preadjust the p H of the solution slightly above 6 to prevent precipitation of the protein. A portion of the protein solution (2 to 5 mg/ml) was placed in the reaction vessel (0.5 ml). After temperature equilibration (10 ~ or 25~ nitrogen was blown over the surface of the solution during the titration. The titration was performed using 0"05 ~-KOI.I standardized with potassium hydrogen phthalate, the solution being constantly stirred with a magnetic stirrer. As a blank, 0.2 M-KC1 solution was also titrated in the same manner. After correction for the blank, titration results were expressed as moles of proton dissociated per mole of protein (Perlmann, 1972). Discontinuous potentiometric titration was also carried out b y adding portions of K O H to the protein solution. The behavior of the protein was the same under these two different procedures of titration. (f) Spectrophotometric measurements Absorption spectra were measured with a Cary 118-C recording spectrophotometer. Fluorescence spectra were recorded on a n Hitachi P e r k i n - E l m e r MPF-3 spectrofluorometer equipped with a corrected spectra accessory. All spectral measurements were made at 25~ with represser concentrations of at least 10 -6 ~. (g) Temperature-jump experiments The equipment used for the temperature-jump relaxation measurements was a combined stopped-flow temperature-jump apparatus constructed in this laboratory (Wu & Wu, 1974). The reaction cell of this apparatus is about 0-3 ml in volume a n d utilizes two conical lenses a t right-angles to each other. A n exciting wavelength of 295 n m from a 200 W I-Ianovia xenon/mercury arc lamp was isolated with a Bausch a n d Lomb grating monochromator. The emission wavelength was isolated from the exciting wavelength by Corning 7-51 a n d 0-52 filters. The changes in fluorescence wore detected with a n E M I 9635QB photomultiplier tube. Signal-to-noise ratios of 1000~3000 were routinely obtained. Temperature j u m p s of 7.5~ were applied to the solution b y discharge of a 0.1 /~F capacitor which had been charged to 10 kV. The resolution time of the apparatus is approximately 10/~s. The temperature-jump was from 17.5 ~ to 25~ in all eases. The kinetic information was recorded on a Tektronix 543 storage oscilloscope and transferred directly on-line to a PDP-11 digital computer b y a Biomation Model 802 transient recorder. The relaxation times a n d amplitudes were calculated b y a least-squares analysis in terms of one or two relaxation processes. The data of successive measurements on the same reaction mixture were accumulated to enhance the signal-to-noise ratio. I n some eases the oscilloscope traces were photographed a n d analyzed graphically. The results were in good agreement with the computer analysis. The experimental u n c e r t a i n t y in the relaxation times was estimated to be a b o u t 10~/o.
3. Results and Treatment o f Data W h e n t h e fluorescence i n t e n s i t y change a t 360 n m was followed, a single r e l a x a t i o n process could be d e t e c t e d w i t h t h e t e m p e r a t u r e - j u m p for solutions c o n t a i n i n g /ac represser i n buffer I ( p i t 7-5) i n t h e absence of inducer. A t y p i c a l oscilloscope trace
462
F. Y.-H.
WU,
P. BANDYOPADHYAY
AND
C.-W.
WU
of the relaxation process following a temperature-jump is given in Figure 1. No relaxation was seen in the absence of the protein. Figure 2 shows t h a t the reciprocal relaxation time, 1]T ~ 13,600 s -z, associated with this relaxation effect is independent of the concentration o f / a c repressor, suggesting the existence of a conformational equilibrium between two different forms of the repressor. kr
R*.
" R.
(3)
1/T = kf +/c~
(4)
kr
The relaxation time for this mechanism
is not dependent on the repressor concentration. I n the presence of the inducer, isopropyl-fl-D-thiogalactoside, two relaxation effects were seen, a fast one in the time range of about 50 ~s and a slow one in the time range longer t h a n 100 ms. The slower relaxation is probably associated with the bimolecular reaction of I P T G and lac repressor, since it occurs in the time scale similar to t h a t of the bimolecular binding of the same concentrations of these molecules observed b y the stopped-flow measurements (Laiken et al., 1972). However, because of its overlap with the optical changes associated with the convection of the solution, this slow relaxation process cannot be reliably analyzed using temperature-jump procedures. The fast relaxation process is essentially the same as t h a t observed in the absence of the inducer. I n addition, the relaxation time for the fast
tl.
