THE JOURKAL OF BIOLOGICAL Ck~ar~srnr Vol. 24i. Xo. 3, Issue of February 10, pp. Sli-924, 102

Printed in U.S.A.

Transport Properties of the Galactose-binding Protein of

Escherichia coli

SFBSTRATE-IXDUCED COSI:ORMLIATIONAL CHANGE*

(Received for publication, August 19, 1971)

WISFRIED Boos, ADRIESNE S. GORDON,~ ROBERT E. HALL, AXD H. DONALD PRICE

From thx Department of Biological Chemistry, Harvard Medical School, and the Biochemical Research Laboratory, Massachusetts Gened Hospital, Boston, Massachusetts 02114

SUMMARY

The galactose-binding protein, a necessary component of a bacterical transport system, is shown by three independent methods to undergo a conformational change upon binding of substrate.

1. A Lineweaver-Burk plot of the binding data obtained by equilibrium dialysis at a protein concentration of 0.4 mg per ml shows heterogenous behavior. Extrapolation of the data at low galactose concentrations ( 10eg to 3 .lO’ M) yields an apparent Z‘&i,s of lo-’ M and at higher galactose concen- trations (3 .lO-’ to lo-” M) an apparent Kdiss of 10e5 M. A Scatchard plot of the binding data extrapolated to high galactose concentrations indicates two binding sites per 36,000 molecular weight.

2. The presence of 10e4 M galactose during analytical poly- acrylamide gel electrophoresis at pH 8.4 results in a changed electrophoretic mobility of the binding protein. This change is also observed in the presence of 1OV M glucose and lop4 M (D-glyceryl)-1-/I?-D-galactopyranoside substrates of the trans- port system. Methyl-1-0-/3-D-galactopyranoside, methyl-l- thio-fl-D-galactopyranoside, and isopropyl-1-thio-o-D-galac- topyranoside at concentrations of lop4 M do not cause the altered electrophoretic mobility.

3. The fluorescence of the protein is increased up to 13.5 % in the presence of substrate between 310 to 350 nm when excited at 290 nm. The increase of fluorescence observed with galactose, glucose, and (D-glyceryl)-1-/3-D-galacto- pyranoside occurs at half-maximal total sugar concentrations of lOW, lo-“, and lo-” M, respectively.

The substrate-induced conformational change does not result in a change of the molecular weight of the protein as measured by high speed equilibrium centrifugation and sieve chromatography through Bio-Gel P-150. The conforma- tional change of the protein cannot be observed by optical rotatory dispersion, circular dichroism, and infrared spec- troscopy. These latter spectroscopic methods show, how- ever, that the galactose-binding protein exists to a large extent in fl conformation.

* This rrorlr was supported by grants from the National Insti- tlltes of Health iGllI-184%. AhI-05507) rind the National Science Foluldation (GK30785X).

+ Present address, Roche Institute of ,\Iolecular Biology, Nut- &, ?;ew Jersey 07110.

Purified binding protein from a transport-negative mutant does not undergo the substrate-induced conformational changes measured by binding, electrophoretic mobility, and fluorescence.

The demonstration (1) that the galactose-binding protein, which was shown to be involved in the function of a galactose- specific transport system in Esckerichia coli (2-5), exhibits two different conformational states prompted further studies of this protein related to its proposed role as a carrier molecule in the transport process.

Models proposed for active transport predict that the carrier molecule exists in two different states, each possessing different binding affinities for substrate (6, 7). As reported previously (l), we did find nonclassical binding beha,vior for the galactose- binding protein. However, those binding data were obtained by ultrafiltration through Amicon filters (8) and were inaccurate because the binding affinit,y appeared to be dependent on pro- tein concentration and the protein concentration was changing during the method of measurement. Determination of the bind- ing affinity by equilibrium dialysis revealed that the substrate, galactose, influenced the binding affinity and, therefore, might be the mediator of the change from one form into t.he other. This was in contrast to our earlier findings (I.), in which the pres- ence of galactose during elcctrophoresis on polyacrylamide gels did not change the strength or position of the two bands ob- tained for the protein. Careful re-examination of the compo- nents of the incubation mixture for polyacrylamide gel clectro- phoresis showed that the omission of sucrose resulted in the appearance of only one band. This suggested that the high concentration of sucrose influenced in a substrate-like manner the relative amounts of the two bands so that the additional presence of galactose appeared to have no effect. This conclu- sion was supported by an earlier finding of Anraku (2) that su- crose in high concentrations inhibited galactose binding. It therefore also appeared likely that the separation of the two forms of purified galactose-binding protein observed with Sepha- dex G-100 chromatography (I) could be due to the influence of the glucose residues of bhe Sephndex on the conformational state

917

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918 Confomational Change of the C;alactose-binding Protein Vol. 247, No. 3

of the proteill. The form with the higher affinity would then be retained to a greater extent than the form with lower affinity, resulting in an underestimation of molecular weights from cal- culations basctl on elution volumes. We now present data confirming the existence of two conformational states of the galactose-binding protein, each possessing different binding affinities. In addition, we demonstrate that the change from one conformational stat,e to the other is induced by substrate.

EXPERIMFZJTAL PROCEDURES

Galactosc-binding Protein-The wild type protein was purified as described previously (1). A mutant galactose-binding pro- tcin isolated from a galactose chemotaxis-defective strain, AM7550 (9)) will be described e1sewhere.l

Sugars-Cr\-stalline cu-D-galactopyranose was obtained from Nutritional l<iochcmicals; ~~glucose, anhydrous, from Fisher; TMG2 from Calbiochem; IPTG and MG from Mann; P-glycerol galactoside was prepared as described previously (LO).

Galactose-binding Activity-Galactose-binding activity was determined by equilibrium dialysis with an apparatus manu- factured bv Interscience, Baltimore, Md. The apparatus con- tains eight double plastic blocks forming eight double chambers separated by a small membrane of dialysis tubing (Fisher). Each individu:rl chamber has a volume of 100 ~1 and contains a small glass bead for sufficient stirring. The plastic blocks are

mounted on a tilted turntable and rotated with about 8 rpm. Opposite chambers are filled with 90 ~1 of galactose-binding pro- tein in 0.01 M Tris-HCI, pH 7.3, and 90 ~1 [l-14C]galactose (50 mCi per mmole, New England Nuclear) or [l-311]galactose (2.86 Ci per mmolc, hmersham) at initial concentrations ranging from 10e8 to 10e4 11. Dilutions were made in 0.01 M Tris-HCl, pH 7.3. The tritiated galactose was purified by paper chro- matography (Whatman No. 3MlL1, phenol-water, 4: 1, w/w). The samples wcrc rotated overnight at 4” and subsequently 50 ~1 were removed from each chamber and counted in 10 ml of Fluoralloy (Beckman).

The excess counts found in the enzyme-containing chamber over the amount in the galactose-containing chamber were re- garded as bound galactose, whereas the counts found in the galactose chamber were regarded as free galactose. The protein concentration was 0.4 mg per ml, corresponding to 1.1. 10M5 M.

At this protein concentration in the range of lO-s to low6 M

final free galactosc concentration, the counts in the enzyme

chamber were about 10 times the amount found in the galac- tose chamber. This ratio decreases with increasing free galac- tose concentration and reaches the value of 2 at 10V5 M free galactose concentration. Measurements at high galactose con- centrations reflect increasing error due to high background. It was therefore necessary to use the relatively high protein con- centration of 0.4 mg per ml since lower concentration of protein would result in even less accurate values at high galactose con- centrations. For the determination of binding activity in the eluate of the Bio-Gel P-150 chromatography, an initial [l-‘“cl

1 W. Boos, manuscript in preparation. 2 The abbreviations used are: TMG, methyl-l-thio-@-D-galacto-

pyranoeide; IPTG, isopropyl-l-thio-P-o-galactopyranoside; MC, methyl-l-0.P-D-galactopyranoside; p-glycerol galactoside, (D-

glyceryl)-1./-n-galactopyranoside (all other sugars mentioned in the text have the D-COnfigUratiOn); ORI>, optical rotatory dispersion; CD, circular dichroism; IR, infrared spectroscopy; V,, elation vollune; V,, void volume.

galactose concentration of 1OV IRI was used. The 1)inding actir- ity of the rnutant binding protein from &rain A\v550 was meas- ured at a protein concentration of 0.3 mg per ml.

Analytical Polyacrylanride Gel Electropdoresis-~lnal?-tics1 polyacrylamide gel electrophoresis was performed in a vertical gel electrophoresis apparatus (EC Apparatus Corp., Philadel- phia, Pa.), using 4-mm thick gel slabs with tight sample slots. The gel consisted of 40 ml of acrylamide solution containing 405; acrylamide and 1.5;; bisxcrylamide (Eastman) in water, 16 ml of Peacock buffer (11) (10.8 g of Tris, 0.925 g of disodium El)TX. 2H,O, and 5.5 g of boric acid per 100 ml of water), 0.16 ml of N, N , N’ , N’- tetramethylethylenediamine, and 94 ml of water; 0.16 g of ammonium persulfate in 10 ml of water was added as catalyst. The acrylamidc solution was deionized prior to use by passing 500 ml of the 40:; acrylamide 1.5:;, bisacrylamide so- lution through a column (1 X 15 cm) of Resyn 300 (Fisher) ion exchange resin, discarding the first 50 ml of cfflucnt. Peacock buffer (10 times diluted) was used as electrode buffer. The samples contained 40 ~1 of 407; acrylamidc solution, 5 ~1 of Peacock buffer, and 5 to 20 ~1 of protein solution containing 5 to 10 pg of protein. The total volume was adjusted to 65 ~1 with 0.01 M Tris-HCl, pII 7.3. The electrophoresis was run for 5 hours at a constant volt,age of 300 volts and a final current of 50 ma. When the electrophoresis was run in t,he presence of sugar, the electrode buffers, as well as the gel slab, contained 10e4 31 final concentration of the sugar used. The gel slab was stained (12) in a solution containing 1.25 g of Coomassie blur (Mann), 46 ml of glacial acetic acid, and 454 ml of 507; methanol for 30 min at room temperature while shaking gently. The stained gels were briefly rinsed with water and destained (12) at room temperature in a solution containing 75 ml of glacial acetic acid, 50 ml of methanol, and 875 ml of water while shaking gently for 1 to 2 days. The destaining solution was changed two or three times daily.

Fluorescerxe Spectroscopy-Fluorescence measurements were performed with the Hitachi-Perkin-Elmer MPF-2A spectro- photofluoromcter equipped with a temperature-controlled cu- vettc-holder attachment. Bn excitation slit of 10 nm and an emission slit of 8 nm were used for the excitation and ernission spectra. All other fluorescence measurements were done with an excitation slit of 4 nm and an emission slit of 10 nm. Es- citation spectra were measured following the emission at 330 nm and emission spectra were obtained by means of an excitation wave length of 290 nm. Measurements of fluorescence as a function of sugar concentration were performed at an excitation wave length of 290 nm and monitoring emission at 330 nm. 111 values reported arc uncorrected. Data were obtained at 24” with a protein concentrat’ion of 16.7 pg per ml (4.6 X 1O-7 MI) in 0.01 M Tris-HCl, pl-I 7.3. In the standard procedure, protein (50 ~1) was added to each of four cuvettes containing 3.0 ml of buffer. The contents were mixed by inversion, allowing at least 5 min for temperature equilibration, and the fluorescence intensity was measured. The sugar was added in a small vol- ume (10 to 100 pl), the contents mixed, equilibrated, and the fluorescence again determined. Since the changes in fluores- cence were often small, a control sample receiving buffer in- stead of sugar was also measured, allowing for dilution and fluc- tuations in the instrument. The results are expressed as per- centage of increase in the fluorescence, corrected for the change observed in the control cuvette. All sugars tested gave zero

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Issue of I’rhruary 10, 19’72 IV. Boos, A. X. Gordon, R. E. Hull, and H. D. Price

FIG. 1. Galactose-binding activity of the galnctose-binding protein as a fujlction of g&ctosc concentratjion measured by eq\lilibrium dialysis. 1 )ouble chttmbers of lOO-~1 volume scp:Lr:Lted hy dialysis t,ltbing were filled with ‘JO ~1 of gtilactose-binding protcia (0.4 mg per ml) in 0.01 >I Tris-I1C1, pll 7.3, and !)O ~1 of [1-14C]- galact ose or [l-311]g:d:tctosc in the s:unc bllffcr. The dialysis was per- formed at 4” for at, least, 12 hours. Fifty microlitcrs from c::lch chamber wcrc counted for radioactivity.

0 I 2 3 4 5 6 7 8 3

I/ Free Galacfose [?/Ad] x IO-7

fluorescence when added to buffer alone. With these precau- tions, it is felt that the error of the measurements is about ~1%.

&o-Gel P-150 Chrolllatography-I3io-Gel P-150 (Bio-Rad) was prepared by stirring the dry beads in sufficient 0.01 M Trjs-HCl, pH 7.3, overnight. After packing, the column (90 x 1.5 cm) TKIS equilibrated with 0.01 M Tris-HCl, p1-I 7.3, for 24 hours, either in the presence or absence of lo-* 31 galactose. A 25.cm pressure head was applied, resulting in R flow rate of 0.06 ml per mill. The protein to be measured was applied in a volume of 1 ml at a concentration of about 1 mg per ml; l-ml fractions 1rei.e collected at room temperature. Ender these conditions, t,he void volume was found to be 30 ml with Blue Dextran 2000 (Pharmac%) The marker proteins, myoglobin, bovine serum albumin, and oralbumin, were obtaiucd from Alarm.

A4101ecular l17eight Determination by Sedimentation Rquilib- 1 iunr;-The molecular weight detc~rmillation by sedimentation eqllilibrium was carried out, as described l)rcviously (1).

Optical Rotatory Dispersion an.d Circular Ilickroism-ORD

and CD spectra were obtaillcd on a Cury 60 spcctropolarimeter with CD attachment. The OR11 npcctra wore obtained at a Ilrotein concentration of 0.40 mg per ml and a path length of 5 mm. The CD spectra wxz obtained at, a protein concentration of 0.55 mg pw ml and :I path length of 1 mm. Mean residue n-eight, of the ~:~lactose-binding protein was calculated to be 126 (2). Galactose, when present, was 10m4 M. This concentra- tion of galacto~c was shown not to contribute either to the CD or ORI> spcctr:t ulldcr the esperimt~ntal conditions.

In.frared Spectroscop?j-The IR sl)cctra were determined with a I’crkill-Elmer spec:trol)hotomtte~, model 202, with t,he use of films, prepared on silver chloride \villdo\vs, which were dried by vacuum desiccation :llter freezing in liquitl nitrogen.

1ZESULT.S

Gnlactosc-binding A Qinity of Two I(‘orms of Galactose-binding Pmfein-Binding of galactose was measured by equilibrium dialysis at 0.4 mg per ml of protein. Fig. 1 shows the Line- I\-caver-Burk plot of the reciprocals of bound versus free galac- tose concentration. Two different slol~s were observed. Ex-

I t I I I I I I I I I I I 1 2 3 4 5 6 7 8 9 lo 11 12 13

Bound Gajocfose / Tofu/ Profein x Free Gohcfose [?/Ml x foe5

FIG. 2. Scatchard plot of the binding data of Fig. 1.

trapolation of the points obt.ained between 3 IO+ M and 1OF M free galactose concentrat,ion yielded an apparent dissociation constant of 1OW M, whereas estrapolation of the values obtained between 3. 10e7 M and 10e5 M yielded an apparent Kdiss of lop5 M.

When the data were plotted according to Scatchard (13), the heterogenous behavior is even more pronounced (Fig. 2). The extrapolation of the curve to high free galactose concentrations indicated that 2 moles of galactose mere bound per mole of galactose-binding protein of 36,000 molecular weight.

The results indicate that the two forms of the binding protein have different binding affinities, and that the relative amounts of the two forms are dependent on substrate concentration. The exact determination of the dissociation constants of both species by this method is not possible due to the fact that the relative amount of protein present in each form at any particular galac- tose concentration is not known. However, the difference be-

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1432412 I4 3 2 4 1’2 m y-9

FIQ. 3. Polyacrylamide gel electrophoresis of galactose-binding protein preparation of a transport-positive and a transport-nega- tive strain of Escherichia coli in the presence and absence of 10e4 M galactose. The gel slabs (4 X 120 X 160 mm) contained 10% acrylamide, 0.375% bisacrylamide, 0.1 M Tris-borate, pH 8.4, and 0.002 M EDTA. The gel shown on the right also contained lo+ M galactose. In this case the electrode buffer also contained 10e4 M galactose. The electrophoresis was run for 5 hours at 300 volts with a final current of 50 ma. The gels were stained in Coomassie blue. The following preparations were applied: 1, Sephadex G-NO-treated shock fluid from the transport-positive strain W3092cy- (ATCC 25939) ; 2, purified binding protein from strain W3092&-; 5, Sephadex &lb&treated shock fluid from the trans- Dart-negative strain AW550: 1. Durified binding motein from strain Aiy550.

, . , * I -

tween the actual dissociation constants at high and low galactose concentration must be even greater than the estimated values.a

The protein isolated from a transport-negative mutant, AW550, also deficient in chemotaxis but still cross-reacting with antigalactose-binding protein antibodies, shows no binding of galactose at concentrations up to 10e4 M.

Analytical Polyacrylamide Gel Electrophoresis of Gulactose- binding Protein-The heterogenous behavior of the binding affiity of the galactose-binding protein indicated that the sub- strate, galactose, acts as an effector in the conformational change of the binding protein. We, therefore, repeated the analytical polyacrylamide gel electrophoresis experiments re- ported in an earlier paper (l), omitting sucrose in the incubation mixture in order to avoid any substrate-like interactions. The necessary density of the sample was obtained by using a 40% nonpolymerized acrylamide solution.

Fig. 3 shows the stained gels of two polyacrylamide gel elec- trophoresis runs at pH 8.4 in the presence and absence of 10-d M

galactose, respectively. The purified wild type protein was compared with crude shock fluid of the transport-positive strain W3092cy- (ATCC 25939), as well as with purified and crude preparations of the transport-negative strain, AW550. In contrast to our earlier findings where the presence of sucrose in the incubation mixture resulted in two bands for the purified wild type protein, now only one band was observed in the pres-

3 At protein concentrations higher than 6 mg per ml, there appears to be a substrate-independent influence of protein con- centration on the binding affinity of the protein. This influence of protein concentration on binding activity, as well as it.s possible effect on the quaternary structure of the protein, are currently under investigation.

WA VEL ENG 7-H i-m/~u/

FIG. 4. Fluorescence spect’ra (uncorrected) of galactose-binding protein in the presence and absence of l@* M galactose and glucose. Galactose-binding protein (16.7 pg per ml) in 0.01 M Tris-HCl, pH 7.3; excitation slit, 10 nm; emission slit, 8 nm. A, excit,ation spectra. Emission wave length was 330 nm. B, emission spectra. Excitation wave length was 290 nm. Temperature was 24’.

ence and absence of 10-d N galactose. However, the position of this one band changed on addition of galactose. The different band positions of the protein with and without substrate in the gel can also readily be seen with rather crude protein prepara- tions. Fig. 3 also shows that the mutant binding protein does not undergo the substrate-induced change in electrophoretic mobility. It can be seen that the presence of galactose itself does not affect mobility since the other proteins in the crude preparations do not show changed positions in the presence of galactose. A series of different, galactose concentrations pres- ent, in both gel and samples showed that the change in band position of the galactose-binding protein occurs, for a constant protein concentration, between 10-G and 5. 10d6 M galactose. At these concentrations the band becomes diffuse, occupying a position between the bands obtained at galactose concentrations lower than 10-G M and higher than 5. lOA M. The effect ap- pears to be specific for sugars which are tightly bound to the galactose-binding protein such as galactose, glucose (14), and /3-glycerol galactoside (3). n-Fucose and MG, sugars for which the protein has a low affinity (3), and TMG and IPTG, sugars which are not measurably bound (3), do not show the change in electrophoretic mobility when the sugars are present at 10V4 M.

Fluorescence of Galactose-binding Protein-The excitation and emission spectra of the galactose-binding protein in the pres- ence and absence of 10e4 M galactose and glucose are shown in Fig. 4, A and B, respectively. The excitation spectrum shows a peak at 288 nm which is increased in intensity by galactose and glucose, whereas the peak position remains unchanged. At more narrow excitation slits the excitation peak is observed to split into a peak at 290 nm and a shoulder at 285 nm. These pre- sumably correspond either to two different tryptophan residues or to tryptophan and tyrosine, respectively. The emission spectra remain unchanged in shape when the protein is excited at 280 or 295 nm, showing that the emission is only due to tryp- tophan (15). The emission spectra (Fig. 4B), show a broad maximum at 340 nm. Galactose increases the intensity and gives a 2 nm blue shift in the emission maximum. The maximum percentage of increase is observed at 330 nm. Glucose also increases the intensity but gives no shift in the emission maxi-

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.ZL.-.LLI - I- . I

,0-s )I , / , I

,()‘i 10.2 10-s 10-C 10-G 10-8

CONCEN JRA J/ON, molar

FIG. 5. Per cent increase in fluorescence of galactose-binding protein versus total sugar concentration. O-0, galactose; m - -m, glucose. Excitation: 290; emission: 330. Excitation slit was 4 nm; emission slit was 10 nm. Protein concentration was 16.7 fig per ml. Temperature was 24”.

TABLE I Total jihorescence increase oj galactose-binding protein in presence

of glucose and galactose

Details are the same as those in the legend to Fig. 5.

Glucose concentration

Total tluorescence increase

Without galactose With 2 X 10-S x gala&se

M % %

0 0 11.3 10-e 3.7 10.9

10-C 5.4 8.2 2 x 10-K 6.4 7.5

10-d 7.1 7.6 10-z 7.7 : 8.2

mum. In contrast to these findings, the spectrum obtained with the mutant galactose-binding protein from the transport- negative strain, AW550, does not show any change upon addi- tion of glucose and galactose up to concentrations of 1O-3 M.

Fig. 5 shows the dependence of the fluorescence increase of the wild type protein (excitation, 290 nm; emission, 330 nm) on the total concentrations of glucose and galactose. It can be seen that galactose shows a maximum fluorescence increase of 13.5%, plateauing at approximately 10e4 ;\I, whereas glucose, even at 10’ M, only shows a fluorescence change of 8.547,. Half-maxi- mal effects are observed at, total concentrations of lo-+ M for both galactose and glucose. However, since at the half-maxi- mal sugar concentrations, the galactose-binding protein (4.6. lo+ M) binds a considerable amount of the total sugar present, the actual half-maximal values are less than those indicated.

The qualitative and quantitative differences in the fluores- cence changes caused by galactose and glucose suggested that the sugars might be bound at different sites. Therefore, the effect of adding both sugars to the same protein sample was tested. Varying amounts of glucose were added to the protein in the presence or absence of 2 x 10v5 1~ galactose. The re-

t I lo-' 10-3 W" 10-z 10-b lo- ' 10-e

CONCEN JRA J/ON, molar

FIG. 6. Per cent increase in fluorescence of galactose-binding protein 0)~~su.s total concentration of various sugars. O-0, MG; O-0, TMG; X-X, IPTG; +M, P-glycerol galac- toside; - - -, galactose. Protein concentration and instrument settings were as in Fig. 5.

sults are shown in Table I. It can be seen that in the presence of both sugars the resultant fluorescence increase was not addi- tive, indicating that the sugars were not bound at two indepen- dent sites. However, from these data it cannot be determined whether they are competing for the same site or whether they are bound to two different but interacting sites.

Fig. 6 shows the effect of other sugars on the fluorescence of of the protein. P-Glycerol galactoside yields a fluorescence in- crease with a concentration of 10m5 M at half-maximal effect. MG, TMG, and IPTG also give fluorescence increases but only at much higher concentrations with half-maximal values of 2. 1o-4 t0 1o-3 M. However, it is not clear whether or not the latter three sugars caused the fluorescence changes or whether the changes were due to impurities of galactose, since approximately 0.1% galactose in the samples would give these effects. All four sugars cause the 2 am blue shift in the emission maximum characteristic for galactose.

Measurements of the time course of the fluorescence increase due to galactose provided evidence that the U- and fl-n-galacto- pyranoses yield different changes in fluorescence of the galactose- binding protein. When crystalline ar-n-galactopyranose is equilibrated with 0.01 M Tris-HCl, pH 7.4, an equilibrium is reached in which 70% of the sugar is mutarotated to the p form. The addition of such an equilibrated solution to the galactose-binding protein result’ed in an immediate change in fluorescence with no subsequent increase as a function of time (Fig. 7). However, when a-n-galactopyranose solutions were prepared and immediately added to the protein, there was an initial fast fluorescence increase followed by a more gradual in- crease which approached the value of the equilibrated sugar. The time course of this change is of the same order as the rate of mutarotation (half-time 25 min). The initial fluorescence increase observed when freshly prepared a-n-galactopyranose is used may be due to the a! form, which then must result in a much lower fluorescence change than the fl form, or it may be due to the effect of impurities of the 0 form in the crystalline LY

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000 5 10 45 20 25 30 ‘5

T/ME, minufes

FIG. 7. Time course of fluorescence increase following addition of galactose. Closed symbols indicate equilibrated galactose; open symbols are a-n-galactopyranose mixed at t = 0. Squares, galactose concentration of 2 X lO+ M; circles represent galactose concentrations of 3 X 10-G M. Protein concentration and instru- ment settings were as in Fig. 5.

.20 -

.I6 -

Mycqlobtn

VI

Gvalbumin

B Cvolbumln

1 80,000

/ - / I

40 60 80

Fraction Number

FIG. 8. Bio-Gel P-150 chromatography of galactose-binding protein in the presence and absence of galactose. The packed column (90 X 1.5 cm) had a flow rate of 0.06 ml per min at a pres- sure head of 25 cm. One milligram of galactose-binding protein was applied in 1 ml of 0.01 M Tris-HCl, pH 7.3. The column was eluted with the same buffer at room temperature and l-ml frac- tions were collected. O-O, absorption at 280 nm; A- --A, binding activity as determined by equilibrium dialysis at an initial [1-Wlgalactose concentration of lo-’ M. Galactose-bind- ing is given in excess counts per min found in the protein-contain- ing chamber over the amount present in the protein-free chamber. This test is not linear since the free galactose concentration de- creases up to lo-fold with increasing protein concentration. The marker proteins, bovine serum albumin, ovalbumin, and myo- globin are run together with the galactose-binding protein and their positions were identified in separate runs. A, chromatogra- phy in the absence of galactose; B, chromatography in the pres- ence of 10e4 M galactose. Before measuring binding activity in the galactose-containing eluate, the fractions were dialysed against 0.01 M Tris, pH 7.3, overnight.

t-‘/2

FIG. 9. Sedimentation equilibrium of galactose-binding protein in the analytical ultracentrifuge in the presence and absence of galactose. High speed centrifugation at 24,630 rpm was per- formed in an AN-D-rotor with a protein sample of 0.52 mg per ml (A) and 0.43 mg per ml (B) in 0.01 M Tris-HCl, pH 7.3, in the presence (B) and absence (A) of lo-* M galactose. The In of the relative fringe displacement is plotted versus the square of the radial distance T from the axis of rotation divided by 2. Experi- ments were performed at 15”.

form. The magnitude of this initial effect is such that it would be given by 7% impurity of the B form.

Molecular Weight Determination of Galactose-binding Protein by Sieve Chromatography on Bio-Gel P-l&)---We reported earlier (1) that the two forms of the galactose-binding protein are sep- arable on Sephadex G-100. In view of the data just presented, the relatively low values obtained for the estimated molecular weights by Sephadex chromatography suggested a possible interaction of the glucose-containing residues of the Sephadex material on both the amount and the elution volume of the two forms of the galactose-binding protein. We therefore repeated the chromatography of the protein with the use of a noncarbo- hydrate-containing molecular sieve (Bio-Gel P-150).

Fig. 8 shows the elution pattern of the galactose-binding pro- tein together with three marker proteins from two columns, one containing 10-d M galactose and the other with no galactose present. Under both conditions, the binding protein elutes as a single symmetrical peak. The estimation of the molecular weight from the elution volume of the binding protein in com- parison to the elution volumes of the marker proteins from plots of V,/Vo versus log molecular weight (16) yields, in both cases, a value of 36,000. This indicates that both conformations of the protein have molecular weights identical with that of the poly- peptide chain as measured by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (1).

Sedimentation Equilibrium of Galactose-binding Protein in Presence and Absence of Galactose-To further study the molecu- lar dimensions of the two forms of the galactose-binding protein, we measured the sedimentation equilibrium profile in the ana- lytical ultracentrifuge in the presence and absence of lOA M

galactose. Fig. 9A shows a representative curve of In c versus r2/2 at an initial protein concentration of 0.52 mg per ml. Fig. 9B represents the same function at a comparable protein con- centration of 0.43 mg per ml in the presence of lO+ M galactose.

In the absence of galactose, the curve is linear, yielding slopes for In c versus r2/2 (17) of 2.55, 2.53, and 2.61 for three consecu- tive runs (Fig. 9A). The presence of 10e4 M galactose also re-

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Issue of February 10, 1972 W. BOOS, A. X. Gordon, R., E. Hall, ad H. D. Price 923

-6003~~ 2X) 230 240 250

FIG. 10. OpGcal rotatory dispersion and circular dichroism of the gnlactose-binding protein. The spectra were measured at room temperature with a Cary 60 spectropolarimeter with CD attachment. A, CD spectrum at a protein concentration of 0.55 rng per ml in the presence and absence of 1OW M galactose (the two curves are superimposible). Path length of the cell was 1 mm. 13, ORD at a protein concentration of 0.4 mg per ml in 0.01 M

Tris-HCl. aH 7.3. in i.he nresencc and absence of 1O-4 M ealactose (the two’k~rves ‘a.re superimposible). Path length was 5 mm. Mean residue weight of the galactose-binding protein was calcu- lated according to the amino acid composition (2) to be 126.

suits iii straight lines with slopes of 2.65, 2.59, and 2.57 for three consecutix,e runs (Fig. 9B). The molecular weight is given by R?‘o/(l - i+)wz where u is the effective reduced molecular weight calculated from the slope of In c versus r2/2 plots, ij the partial specific volume, p t,he density of the solvent, and w the angular velocity. With a ij of 0.73 (2) and an average n of 2.58, the calculated molecular weight is 34,000, representing the molecu- lar weight for both forms of the prot,ein.

Optical Rotatory Dispersion, Circular Dichroism, and Infrared Spectroscopy of Two Forms of Calactose-binding Protein-The large difference in the migration of the two forms of the galactose- binding protein on polyacrylamide gels suggested that they might have different ORD, CD, and IR spectra since these methods are frequently used to distinguish different conforma- tional states of proteins (M-20). Fig. 10 shows the CD and ORD spectra of t,he protein in the presence and absence of galac- tose. The salient features are (a) an ORD trough near 230 nm and a cross-over below 220 nm and (1,) a CD band at 219 nm, a shoulder near 20X iim, and a cross-over at 203 nm. These spectral properties are iudicative of p structure (E-20). Since there are no characteristic a-helical bauds, a low o-helix content, probably less than lo”:, would be predicted.

The infrared spe&um of the galactose-binding protein in the presence and absence of galuctose in comparison to that of albu- min is shown in Fig. 11. The galactose-binding protein exhibits under both conditions, in contrast to albumin, a strong band at 1635 cm-l which has been attributed to /I conformation (21, 22). h quantitative estimation of the amount of /3 conformation from the ORD and CD spectra is not possible since it has been shown that the position and rotatory strength of the bands characteristic of p conformation are side chain-dependent (18). However, the addition of galactose at a concentration that has

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G .05 -

z .04-

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8 .02-

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L I L 1600 1700 1800

FIG. 11. Infrared spectroscopy of galactose-binding protein in the presence and absence of galactose. Twenty-five microliters of galactose-binding protein solution of 1.5 mg per ml, in 0.01 M

Tris-HCl, pH 7.3, in the presence and absence of 1OW M galactose was frozen in liquid nitrogen on silver chloride windows and freeze-dried. 1, no galactose; 2, 1OV M galactose; 3, bovine serum albumin.

been shown to cause a conformational change has no effect on the ORD, CD, and IR spectra of the galact.ose-binding protein.

DISCUSSIOX

Several lines of evidence indicate that the galactose-binding protein is a necessary component of a specific galactose trans- port system in E. co& the /3-methylgalactoside transport system (2-5). We recently reported that this protein occurs in two different conformational states (l), a property which has been postulated for carrier proteins (6, 7). In t,he present publica- tion we demonstrate the effect of substrate on the two conforma- tional states which can be directly observed by measurements of binding affinity, by electrophoretic mobility of the protein on polyacrylamide gels, as well as by fluorescence of the protein.

The dat,a indicate a conformat’ional change in the protein molecule upon binding of substrate since both forms have iden-

tical molecular weight as measured by analytical ultracentri- fugation and molecular sieve chromatography in the presence and absence of galactose. The substrate-induced conforma- tional change is specific in the sense that only sugars which are bound to the protein cause the shift in electrophoretic mobility as well as the increase in fluorescence. The only apparent anomaly is XG. This sugar has been reported as a substrate for the fl-methylgalactoside transport system (23). However, binding of MG to the galact,ose-binding protein could only be demonstrated indirectly by its weak interference at high con-

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924 Conj’omational Change of the Galactose-E&&.diny Protein Vol. 247, No. 3

centrations with galactose binding actirity (3). This indicates that the binding protein has a low affinity for MG. Indeed, the data on MG inhibition of galactose uptake in W3092cy- (ATCC 25939) (3), the strain from which the wild type binding protein was isolated, indicate that the apparent Ii, for MG uptake must be higher than 10e4 N, in disagreement with the value obtained by Rotman ef al. (2 x 10M5 M) (23) measured in strain S151. Therefore, ironically, MG does not appear to be a good substrate for the /?-methylgalactoside transport system in w3092cy-.

A nonclassical binding behavior for the substrate galactose has been demonstrated at a protein concentration of 0.4 mg per

ml, which we attribute to the change in conformation upon binding of substrate. When the binding data are plotted according to Scatchard (13), the extrapolation to high substrate concentration shows that 2 moles of glactose are bound for 1 mole of protein, assuming a molecular weight of 36,000. The pattern seen on the polyacrylamide gel electrophoresis in the presence and absence of substrate would suggest that under each of these conditions only one form is present. This implies that binding induces the change in conformation very much in anal- ogy to a proposed induced fit mechanism (24). Binding of one molecule of substrate should then result in a conformational change yielding an additional binding site for a second substrate molecule with a lower affinity than the first site.

The substrate-induced conformational change has also been demonstrated by fluorescence measurements. Again, one ob- serves that only substrates of the transport system are effective at low concentrations in producing the change, whereas structurally related sugars which are not transported by the system show no change at these concentrations. The data do not permit spe- cific assignment of the fluorescence changes to either tyrosine or tryptophan residues but they do indicate a difference between the conformational changes induced by glucose and those in- duced by galactose.

The substrate-induced conformational change of the galactose- binding protein is not large enough to result in significant differ- ences in CD, ORD, and IR spectroscopy. However, these spec- troscopic measurements did reveal that the galactose-binding protein is present in predominantly /3 conformation. Penrose et al. (25) have reported similar CD and ORD spectra for the leu- tine-binding protein, although the spectra were not interpreted as such. Indeed, /3 conformation could explain the prolate ellip- soid shape of the sulfate-binding protein as observed by Lang- ridge et al. (26).

We propose that the substrate-induced conformational change of the galactose-binding protein is related to the mechanism by which galactose is accumulated through the osmotic barrier of the bacterium. since t)he bindinn urotein isolated from a trans-

“ I

port-negative mutant does not show this change. The relation- ship between the conformational change of the protein i~z vitro to its supposed function ilz viva, as well as the relationship to the energy consumption necessary for the accumulation of substrate against a concentration gradient, is at present only subject to speculation. More has to be learned about the interact,ion of the binding protein with the cytoplasmic membrane to approach this problem.

Acknowledgments-We wish to thank Dr. Herman M. Kalckar for his generous hospitality and his encouragement during t’his work. We are grateful to Dr. Joseph Avruch for his aid in ob- taining the IR spectra and to Xss Lena zUberico for excellent technical assistance.

1. BOOS, W., .ISD GORDOX, A. S. (1971) J. Biol. Chem.., 246, 621. 2. ANRAKU, Y. (1968) J. Biol. Chem., 243, 3116,3123, 3128. 3. Boos, W. (1969) Eur. J. Biochem.. 10. 66. 4. Boos; W., END S~nvas, M. (1970) Eur. J. Biochem., 13, 526. 5. LENGELER, J., HERIMNS, K. O., UNS~~LD, H. J., AND Boos, W.

(1971) Eur. J. Biochem., 19, 457. 6. Foi, Cl F., AND KENSED<, E: P. (1965) Proc. Nat. AcacZ. Sci.

U. S. A., 54, 891. 7. WINI~LER, H. H., MD WILSON, T. H. (1966) J. Biol. Chem.,

241, 2200. 8. 9.

10.

PAULUS, H. (1969) Anal. Biochem., 32, 91. HAZELBAUER, G., MJD ADLER, J. (1971) AVature New Biol., 230,

101. Boos, W., LEHMMS, J., .ISD WALLENFELS, K. (1966) Cc&o-

hyd. Res., 1, 419. 11. PEACOCK, A. C., BUNTISG, S. L., AXD QUEEN, K. G. (1965)

Science, 147, 1451. 12. 13. 14. 15.

16. 17.

WEBER, K., AND OSBORN, M. (1969) J. Biol. Chem., 244. 4406. SCATCH~RD; G. (1949) An&. N.‘ Y. &ad. Sci., 51, 660. ANRAI~U. Y. (19671 J. Biol. Chem.. 242. 793. UDENFR&ND,~ S. ‘(1962) Fluoresckzce ‘assay in biology and

medicine, academic Press, New York. ANDREW~, I’. (1964) Biochem. J., 91, 222. ROARK, D. E., BND YPHANTIS, Il. A. (1969) Ann. N. Y. Acacl.

Sci., 164, 245. 18. STEVENS, L., TO~XEND, R., TIMASHEFF, S. N., FASM~N, G. D.,

AND POTTER, J. (1968) Biochemistry, ‘I. 3717. 19. 20.

BEYCHOK, S. (i968) Ann. Rev. Biochk, 37, 437. YANG, J. T. (1967) in G. FASMAN (Editor), Polycr-amino acids,

p. 239, Marcel Dekker, New York.

REFERENCES

21. SUSI, H., TIMASHEFF, S. X., AND STEVENS, L. (1967) J. Biol. Chem., 242, 5460.

22. TIMASHEFF, S. X., SUSI, H., AND STEVENS, L. (1967) J. Biol. Chem., 242, 5467.

23. ROTMAN, B., Gan-~sas, A. K., AND GUZMAN, R. (1968) J. Mol. Biol., 36, 247.

24. CONWAY, A., ASD KOSHL~ND, D. E., JR. (1968) Biochemistry, 7, 4011.

25. PENROSE, W. R., Z~ND, R., an-~ OXENDER, D. L. (1970) J. Biol. Chem., 245, 1432.

Science, 169, 59. 26. LANGRIDGE, R.., SHINAGS~T.I~, H., AKD PARDEE, A. B. (19iO)

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Winfried Boos, Adrienne S. Gordon, Robert E. Hall and H. Donald PriceSUBSTRATE-INDUCED CONFORMATIONAL CHANGE

:Escherichia coliTransport Properties of the Galactose-binding Protein of

1972, 247:917-924.J. Biol. Chem. 

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