Microcalorimetric study of glutamine fixation on the glutamine-binding protein of Escherichia coli

Vol. 86, No. 4, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BIOCHEMICAL

February 28, 1979

Pages 1118-1125

MICROCALORIMETRIC

STUDY OF GLUTAMINE FIXATION

ON THE GLUTAMINE-BINDING

B. Marty,

C. Gaudin,

PROTEIN OF ESCHERICHIA COLI

M. Ragot,

Laboratoire

A. Belaich

de Chimie

13274 MARSEILLE Received

January

and J.P.

Bacterienne,

Belaich

C.N.R.S.

CEDEX 2, France

6‘1979

SUMMARY The enthalpy variation (AH) induced by addition of glutamine to glutamine binding protein isolated from E.coli has been studied by microcalorimetry. The reaction was very exothermimfree energy variation (AG) was calculated from the dissociation constant (KU) measured by dialysis techniques. The entropic variation (AS) was deduced from AG and AH values ; it was found highly negative,indicating that an important conformational change is occuring. Comparison with others binding proteins and possible significance of such a phenomenon is discussed. INTRODUCTION Concerning about

gram negative

the periplasmic

mode by which Several

they

binding

cytoplasmic

proteins

do transfer

investigators

assumed

membrane

may be preceded

of the binding

protein

Boos (4)

Evidence ted

of E.coli

typhimurium

;

. These

fluorescence,optical sis,

denaturants

: Galactose Sulfate studies rotatory

involved ligand

that

a direct step.

thus

(5)

Some authors induced

recently

(6,7), (11)

Maltose

(8)

and Histidine

have been done utilizing dispersion,

1118

the exact

membrane

(1,2,3).

interaction did

suggest

with that

conformational

reviewed

this

several

I . R spectroscopy,

change for

subject.

has been presen-

and Glutamine binding

the

such

necessary

changes

(12)

questions

is

stereochemistry

of such conformational

and N.M.R.

@ 1979 by Academic Press, Inc. in any form reserved.

of reproduction

cell

and specific

the right

0006-291X/79/041118-08$01.00/0 Copyright All rights

in transport

across

by a substrate

and Singer

in support

in a few cases

proteins

creating

one of the outstanding

their

is a necessary

an interaction

the interaction.

bacteria,

(9,lO) proteins methods gel

binding of 2. like

electrophore-

BIOCHEMICAL

Vol. 86, No. 4, 1979

Recently valine

binding

protein

In the

present

paper

that heats cal

we studied

binding

(LIV-BP)

of E.coli

we report

was done upon glutamine of binding description

hypothesis

and affinity

a strong

results

of ligands

on the leucine,

isoleucine

by the mean of microcalorimetry obtained

binding

protein

constant,

of the binding

that

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

during (Gln-BP)

parameters

conformational

change

same kind

of E.coli. that

The results

reaction.

the

occurs

permit

obtained

,

(13). of study

We measured a thermodynamisupport

upon the binding

the reaction.

MATERIAL AND METHODS E.coli cultures were grown Lerner and Lin (14)

: Strain Gln PI was a kind gift of D. Leon A. Heppel. by Tanaka, in a synthetic medium described previously supplemented with 1% sodium succinate.

All

Chemicals : Isotopic material (L G-3H, Glutamine, L U-14C Glutamine) was obtained from the Radiochemical centre Amersham. Non-radioactive L + Glutamine was purchased from Fluka. DEAE cellulose was from Whatman, Ultrogel from L.K.B. and scintillation liquid from Lumac. All others chemicals were obtained from Prolabo and Merck. Protein

preparation

:

The Gln-BP has been purified to homogeneity by a method derived from the one published by Wiener and Heppel (9). One hundred grams (wet weight) obtained from a 16 liters culture (Chemap fermentor) were osmotically shocked. The four liters crud shock fluid was concentrated by ultrafiltration throught an Amicon UM 10 membrane. The filtrate, after extensive dialysis against 10 mM Tris HCl pH 7,4 buffer, was applied on a column (26x700mm) of DEAE cellulose. The Gln-BP was not retained and This material was then concentrated appeared ahead in the flow-through fraction. and chromatographed on an Ultrogel ACA 4/4 column (26x1000 mm) equilibrated with 10 mM pH 7 phosphate buffer containing 0,lM NaCl and 5 mM sodium azide. The Gln-BP was then denatured by dialysis against an Urea 8 M solution and renatured by dialysis against the same buffer as indicated above. Binding

assays

:

'The glutamine binding activity was determined by two different methods : equilibrium dialysis and rapid dialysis. Equilibrium dialysis was carried out in Lucite chambers at 25'C as previously described by I-urlong and Wiener (15) and by Wiener and Heppel (9). Rapid dialysis was done according to the technique of Colowick and Womack (16). Calorimetric

Measurements

:

Heats of binding of glutamine to Gln-BP were measured at 25 + 0,l'C using an LKB batch calorimeter 10700-2and an LKB flow calorimeter 10700-l-. In both cases, a Keithley 150 B microvolt ammeter coupled to a Sefram recorder were used to amplify and record output from the calorimeter. The electrical calibration heaters were checked by measuring the precision of the heat of neutralisation of HCl 3.10-4N.

1119

Vol.

86,

All

No.

4, 1979

binding

BIOCHEMICAL

experiments

were

AND

performed

BIOPHYSICAL

using

RESEARCH

the

COMMUNICATIONS

10 UV output

of the amplifier.

Concerning the batch calorimeter, all heats of reaction, substrate and protein dilution were determined in separate experiments in order to avoid errors due to differences in response of the thermopiles in each cell. Wet glass cells were used for all experimental work. Final volume in each cell was 6 ml.

In the case of the flow calorimeter, pumps speed was adjusted to 10 ml per hour For all binding experiments the protein pulse volume was precisely fixed to 1 ml. Protein

concentration

was determined

by the method

of Lowry et al

(17). RESULTS The thermograms figure

during

the

binding

reaction

are

shown in

I.

Binding

of glutamine

to Gln BP

have been measured All

obtained

the results

dilution

are

upon protein are

(19)

during

those

to the work

in order

employed

was treated

Then

and /or

same order

rapid

all

process.

Heat values

concentrations.(Table

and the heats

of protein

I).

and substrate

to the binding

the dissociation As it

when compared

by the binding

conditions

The value

calculate

we did

while

(KD) measured

can be seen,

its

indicated

capacity

were

obtained

for

protein by

the same as KD is of the

in the litterature.

reaction

constant

for

study

renatured,as

retained

Experimental cell.

and to a recent

8 M and then

was checked

dialysis.

encountered

(18)

the glutamine

the Gln-BP

From the thermograms

negative

of varied

by urea

in the calorimetric

as those

associated

exothermic

of Amanuma et al

to release

purification.

equilibrium

sol' utions

in good accordance

the Gln-BP

in methods,

a strongly

negligible.

According we did

is

the

free

by dialysis.

the enthalpy

to the values

the enthalpy energy

variation

(AG) was deduced

Data are

variation

upon binding

obtain

in a previous

we did

(AH)

shown in table is

strongly

study

of

LIV-BP (13). Also of Gln-BP and for

for that

strongly its

ligand.

reason

it

negative,

the values

The entropy is

the most

variation,

interesting

1120

of AG reflect

from

the high

affinity

AS, is very highly

negative

datum to be discussed

below.

I.

Vol. 86, No. 4, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3

II&,,

0

w i

$I&;,, ”

c t

of binding reaction. Fig. - 1 - Thermograms 1,2,3 : Flow calorimeter experiments (Amplifier 10 yV- Recorder 50 mV). : heat of dilution of Gln-BP (Binding sites = 0,027 pmole) la lb : heat of reaction of Gln-BP with glutamine. x 2 2 a and 2 b : idem but Gln-BP concentration 0 3 a and 3 b : ' x4 2 c : heat of calibration corresponding to 5,06 m Joules (1,210 meal) 4 : Batch calorimeter experiment (Amplifier 1OuV - Recorder 100 mV1 a : heat of dilution of Gln-BP (Binding sites = 0,141 vmole) II I, Glutamine a': of Gln-BP with Glutamine b: heat of reaction c : heat of calibration corresponding to 11,21 m Joules (2,68 meal).

1121

0,141*

0,108

4 b

3b

2b

lb

Thermogram number (cf Fig 1)

2

2

3

2

Table I : fixation

2 .!352

2.305

1.36

0.571

12.34

3.63

5.68

2.39

,

- 21

- 21.5

- 25.4

- 21.2

Kcal.mol-'

AH

upon Glutamine

Q (mean value) m cal " Joules

glutamine

Number of experiments

parametersof AG

protein.

II

II

"

- 9.13

Kcal.mor'

Binding

- 39.8

- 41.5

- 54.6

- 40.5

Cal.mor!

AS

c1

(9,15,16).

Experimental

The dissociation

was 25 2

was calculated

- AS value

temperature

was deduced

0,l"C.

=-RT

Ln l/KD

previously

T

: AS = AH - AG

AG

techniques relationship

to the

to the equation

the following

according

according

from

(KQ) was measured

- AG value

constant

published

In the flow calorimeter experiments the quantity of ligand was 5 pmoles per ml and in the batch calorimeter experiments (*) the quantity of ligand in the microcalorimetric cell was 2 pmoles i.e. in both cases largely in excess when compared to the number of binding sites.

II

II

0,054

O,2

0

0,027

PM

b

Binding Siters pmole

Thermodynamic

Vol.

86, No.

BIOCHEMICAL

4, 1979

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

a full

thermodynamic

DISCUSSION The three description

of the

obtained This

parameters

it

binding

calorimeters

in spite

molecules

near

suggest

that

important only

binding

the binding

and Heppel

of glutamine

of the molecule. were

conformational

the

binding

they

burying

to Gln-BP

in their

could

was entro-

a disorganization

of AS obtained

is

accompanied

the entire

by an

molecule

fluorescence

an not

of Gln-BP

of the two tryptophan

residues

whether

or whether

the tryptophan

change

study

not conclude

by glutamine

hypothesis

conformational

residueg.

the tryptochans glutamine

Results

caused

found

if we assume that

in the first

would

weak since

be probably

that

a

in this hypothesis

limited

to the

site. Kreishman

indicating binding

the reaction

values

may concern

found

and hidden

the second

resulting

negative

the environment

time

site

change

support

that

(9)

changed

At that

at the active

study

that

completely

site.

Wiener addition

and the batch

was undergoing

of Glutamine

change

the flow

driven.

site.

case the highly

conformational

is enthalpy

AH and L!S values

; we concluded upon binding

reaction

From the values

of the later.

we found

the binding

In the present

from

sensivity

(13)

here

the protein,

formation

obtained

of the lesser

obtained

and that

of water

the complex

upon LIVBP

from those

py driven

that

allow

have been determined.

by the results

In a study distinct

reaction

can be deduced

is demonstrated

(AG, AH, hs) that

that

et al

the protein

and that

the

after

binding,

would

be associated,

tion.

Shrake

et al

too,

hand,

(20)

were

highly

the protein is

concluded

was undergoing

tryptophans

On the other that,

(10)

negative

in a more ordered studying

a large

buried

ligand

glutamine

1123

their

PMR study

of Gln-BP

conformational

change

at the same time values

complex, state

than

synthetase

.

of AS allow

including the

us to say

water free

upon

molecules

protein

(GS) of E.coli

that

in solufound

a

Vol.

86,

No.

very

4, 1979

similar

BIOCHEMICAL

result;their

in a more ordered

conclusion

state

than

Conformational suppose

that

tion

of a Gln;BP-glutamine

side

and water

AS variations

were

Arabinombinding ATPase

and entropic

reaction

emphasis

these

point

on the fact as temperature

GS-glutamine

variation

occuring

of AS lead

may result

all

around.

out

that

such large

authors

: Schleif

(21) the

with

and by Belaich

et al

(23)

in their

upon

along

of view.

these

independent

work

the enthalpic

temperature

dependent.

of biochemical

and that

this

negative

(Na+,

K')

study

of

the

Mprotein

show that

enthalpic

when considered

variation

made the

We should

like

reactions last

out-

who studied

but opposite

cases

kinds

large

permease

the present

in absolute,

In these

and so

with

Bgalactoside

forma-

oriented

or very

(22)

binding

is

us to

into

residues

et al

are large,

that

the complex

Kuriki

exemples,

favorable

COMMUNICATIONS

some hydrophobic

by different

of E.coli,

contributions

from energetic

thought

observed

and lactose All

with

can be pointed

RESEARCH

alone.

change

reorganized

of the electriceel

of E.coli.

was that

and very negative

complex

protein

Thiodigalactoside

binding

it

also

the complete

molecules

BIOPHYSICAL

the protein

change

upon binding

Moreover

AND

concept

are

to lay

generally

must now be

reconsidered.

- REFERENCES -

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Berger, E.A. and Heppel, L.A. (1972) J. Biol. Chem., 247, 7684-7694. Kaback, H.R. (1970) Ann. Rev. Biochem., 39, 561-598. Oxender, D.L. (1972) Ann. Rev. Biochem., 41, 777-814. Boos, W. (1974) Ann. Rev. Biochem., 43, 123-146. Singer, S.J. (1974) Ann. Rev. Biochem., 43, 805-833. Boos, W., Gordon, A.S., Hall, R.E. and Price, H.D. (1972) J. Biol. Chem., 247, 917-924. Silhavy, T.J., Boss, W. and Kalkar, H.M. (1974) in Biochemistry of Sensory Fuctions, Jaernicke. L. Ed. Berlin, Springer-Verlag, 165-205. Szmelcman, S., Schwartz, M. , Silhavy, T.J. and Boos, W. (1976) Eur. J. Biochem., 65, 13-19. Wiener, J.H. and Heppel, L.A. (1971) J. Biol. Chem., 246, 6933-6941. Kreishman, G.P., Robertson, D.E. and Ho, C. (1973) Biochem. Biophys. Res. Commun., 53, 18-23. H. and Pardee, A.B. (1970) Science, 169, 59-61. Langridge, R., Shinagawa, Robertson, D.E., Kroon, P.A. and Ho, C. (1973) Biochemistry, 16, 1443-1451.

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Vol. 86, No. 4, 1979

BIOCHEMKAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

J.C. and Belatch, J.P. (1977) 13. Gaudin, C., Marty, B., BelaTch, A., Sari, Biochem. Biophys. Res. Commun., 78, 377-382. 14. Tanaka,S,, Lerner, S.A. and Lin, E.C.C. (1967) J. Bacterial., 93, 642-648. 15. Furlong, C.E. and Wiener, J.H. (1970) Biochem. Biophys. Res. Commun., 38, 1076-1083. 16. Colowick, S.P. and Womack, F.C. (1969) J. Biol. Chem., 244, 774-777. 17. Lowry, D.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) J. Biol. Chem., 193, 265-275. 18. Amanuma, H., Itoh, J. and Anraku, Y. (1976) J. Biol. Chem., 79, 1167-1182. 19. Gaudin, C., Marty, B., Ragot, M., Sari, J.C. and Belaich, J.P. (1978) Biochimie, 60, 353-359. 20. Shrake, A., Powers, D.M. and Ginsburg, A. (1977) Biochemistry, 16, 4372-4381. 21. Schleif, R. (1969) J. Mol. Biol., 46, 185-196. 22. Kuriki, Y., Halsey, J., Biltonen, R. and Racker, E. (1976) Biochemistry, 15, 4956-4961. 23. Belaich, A., Simonpietri, P. and Belaich, J.P. (1976) J. Biol. Chem., 251, 6735-6738.

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