Vol. 88, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSKAL

RESEARCH COMMUNICATIONS

May 14, 1979

Pages 124-129

COMPARISON TRYPTIC

OF THE CORE

M.

LAC

BY HYDROGEN

EFFECT

J. Ramstein,

E. COLI

REPRESSOR

EXCHANGE

OF INDUCER

Charlier,

J. C.

AND

ITS

STUDIES.

BINDING

Maurizot,

A. G. Szabo

t

and

C. Hkl’ene lA,

Received

January

Centre de Biophysique MoMculaire avenue de la Recherche Scientifique 45045 Orldans Cedex (France) 22,1979

Summary Using the hydrogen exchange technique we have compared the E. Coli Lac repressor and its tryptic core. The effect of isopropylb -D-thiogalactoside (IPTG) binding has also been examined. Within the limits of experimental errors the hydrogen exchange curves for both the repressor and the core are the same. This supports the conclusion that “headpieces” (N-terminal fragments released by limited tryptic hydrolysis) and core are rather independent structural units. The binding of IPTG to the core does not affect its hydrogen exchange curve whereas the binding of IPTG to the repressor results in the blocking of a least ten protons. This result demonstrates that the headpiece is involved in the conformational change induced by the binding of IPTG. Introduction Lac

repressor

controls

the expression

to the -lac operator. Sugarslike allolactose galactoside (IPTG) act as inducers of the operon

of the lactose

binding

releasing

it from

and for

the operator

review

and discussion

experiments are

repressor

conditions

digestion

located

see ref.

of experiments

sin on lac

by binding

by

b -D-thio-

to the protein

and

the DNA.

Genetic

the results

or isopropyl-

operon

ofl:the

-lac

+ A. G. S. is a visiting scientist : Division Permanent address Canada KIA OR6.

This

restricted repressor

that

the binding

sites

regions

within

the protein

(for

supported

by

action

of tryp-

conclusion

a biochemical

is also

approach.

124

The

for

and under

appropriate

experimental

produces

a mixture

of monomeric

under the CNRS-NRCC of Biological Sciences,

0006-291X/79/090124-06$01.00/0 Copyright @ I979 by Academic Press, Inc. AN rights of reproduction in any form reserved.

shown

on different l-2).

using

is very

have

IPTG

exchange programme. N. R. C. C., Ottawa,

Vol. 88, No. 1, 1979

amino-terminal

fragments

and

I-59

together

ric

core

has

binding et

that

full

very

likely

well

as

ted

within

in

specific

the

tetrameric

is

within

still

the

whose

for

dichroism

and

evidence which

for might

between cer

of

Materials

of we

its

repressor

of the

core

devoid

any

shown

tetrame-

detectable

in

the

(5). non

binding

DNA by

to DNA

involved

l-51

The

recently

binds

inducer

residues

(3).

of

been

is

can fact

protein

explanation the

is

has here core.

rules

out

that

IPTG

Jovin

It is

thus

specific

activity

as

is

of

not

the

of the been

effect

been

protein

exchange of

IPTG

an

loca-

one

absorption, used

in

order

to

changes

and In study has

the

lo-

of the been

provide

are

seen

correlation allosteric

connection

also

with

circular

a clear

repressor

effect. effector

a new

structure

binding

located

allosteric

Although

(HX)

are

opera-

competition

to

like

(6-9).

lac

direct

is

demonstrated.

a hydrogen The

any

of the

sites

protein

have

changes

binding

binding

techniques

spectroscopy

still

the

both

of the

Several

change

influence

that

conformation

a versatility

IPTG

is

whereas

of IPTG

conformational

report

and

lac

binding

fluorescence

t ryptic

it has

region

operator.

reflect

effect

hand,

The

a conformational

problem and

the

such

but

coli

binding

switches

affinity

activity

for

core.

plausible

binding

wer

DNA

regions

most

other

understood.

different

Thus

the

of E L-

the

poorly

accounting tetrameric

amino-terminal

the

How tor

On

the

“headpieces”

binding

headpiece

that

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a trypsin-resistant

inducer (4).

the

called

with

activity

al.

BIOCHEMICAL

indu-

with -lac

this

repressor examined.

Methods

lac repressor from strain BMH 493 was purified accor--E. coli ding to the method described by Muller-Hill et al. (14). Tryptic digestions were performed in 1 M Tris-HCI (pH 7. 5) 30 % glycerol. The amino terminal segment and the core were separated according to Geisler and Weber (15). Concentrations of lac repressor and core were determined from UV absorption measurementsing c 280 = 21,400 and 16,600 per protomer for the lac repressor and the tryptic core, respectively. The tritium Sephadex exchange experiments were carried sium phosphate pH 7.25, 0. 1 mM

method of Englander out at 0°C in KPD dithioerythritol (DTE)

was used (16). All buffer (0.2 M potas). Exchangeable

hydrogen sites were labelled with tritium by incubating 0. 5 ml of the protein solution (0. 5 ml, 2~10~~ M protomer) with tritiated water (10 ~1, 1 Ci/ml) for 16 hours and a half. To initiate the exchange of protein-bound tritium the sample was passed through a Sephadex column (G 25 fine grade, 1 cm x 7 cm) This column removed the free tripreviously equilibrated with KPD buffer. tium and the protein peak was collected in a test tube. In order to measure the amount of tritium still bound at any given time t aliquots of 250 pl were

125

Vol. 88, No. 1, 1979

BIOCHEMICAL

passed through a with KPD buffer. time t. The protein the concentration ber of hydrogens ted from the ratio

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

second Sephadex column (G 25, 1 cm x 4 cm) equilibrated This column removed the tritiated water formed during peak was collected in four test tubes and for each tube of protein and the radioactivity were measured. The numper protomer remaining unexchanged at time t was calculaof tritium counts per protomer (16).

Radioactivity was measured by pipetting 0. 5 ml of the aqueous samples into 5 ml of liquid scintillation counting mixture (BRAY) and counting in an Intertechnique SL 30 spectrometer. Protein concentrations were determined from fluorescence intensity measurement. Fluorescence intensities were measured with a Jobin Yvon spectrofluorimeter modified to corWhen complexes with IPTG were inrect for fluctuations in lamp intensity. vestigated experimental conditions were chosen to ensure saturation of the IPTG binding sites during the whole exchange of the protein bound tritium. For this purpose the first and second columns were equilibrated with KPD buffer containing 2 mM IPTG and 0. 1 mM IPTG, respectively. Results

and

Discussion The

absence

as

following

there

of its

as

is

tryptic

in

the

does

in

the

general

and

in

hydrogen

kinetics

and

are

the

HX

of

the

the

its

the curves HX

exchange

on

limits

curves of

these

results

is

in

1.

The

the

repressor

and

repressor

and

shows

of

repressor

-lac

IPTG

core

experimental

of -lac

behavior

&

Fig.

of

of the

presence

of the

tryptic

presented

i) within

comparison

aspects

on

hydrogens posed

discussing

the

that

core

slowed

the

accessibility

are

exposed

peptidic

perature they

do

hydrogens

not

are

IPTG

whereas

down

down

exchange

by

protein

exchange

with

grossly

protein

to of

upon

the

solvent

solvent

by

six peptidic

half

times

in

the

curves

in

internal

orders

and

on

structural very are

the

of fig.

1.

hydrogen of

magnitude are

extent

bonds (11). very

(10). our

time

the their It has

pH

exchange

to

these

tem-

(11)

and these

rate

shown the

ex-

and

when

been

de-

Freely

range

contrary

sensitive

rates

to which

under

second On

Peptidic

differing

the

and

in

hydrogens

protein.

fluctuations

rapidly

briefly

is

that

the

conformation

(10). As

core

of

their

one

rates

recalling

a structured

exchange

involved

worth

in

to

appear

is

exchange

hydrogens

conditions

it

of hydrogen

of a structured

pending

merit

IPTG

between

the

exchange

hydrogens

of the

:

made

repressor

binding.

some

wed

be

the

affect

-lac of

absence

Before

thus

the

presence

difference ; ii)

not

hydrogen

IPTG

no

core

for

can

core

tryptic

binding the

well

curves

observations

errors

its

HX

have

we

have

identical

already HX

mentioned curves.

Unless

126

the

-lac there

repressor are

unlikely

and

the

tetra-

compensa-

slo-

Vol. 88, No. 1, 1979

BIOCHEMICAL

I I

Figure for the

I 2

1 : Absolute lac repressor

unexchanged (o), the

core

and the complex IPTG-core pH 7.25, 0. 1 mM dithioerythritol Temperature 0°C.

ting

effects

celled with

by

the

the

and

sixty significantly

exchange

too

placed

by

16 hours

the

“headpiece”

-lac

repressor

the

stability

must

have into

fast

tritium

for

the

their

amino

and

of

exchange

the

hydrogens

HX

because

in

the

at no

the

In

core

a few core

hydrogens

are

technique.

Thus

the

in other

structure points

they

then

Our

role

molecule.

only

exchange

0°C.

-lac

data

and

expected that

be

easily

to

exchange

the

modified

the

“headpieces”

seem

to the

of

“headpiece”

“headpieces”

rapidly

hydrogens

the

excision not

the

of contri-

not

re-

in

‘Hz0

presence

the

rest

not

contribute

d

of the

“headpieces”

solvent

la-

they

were

does

If the

too

127

structure

can-

e. g.

incubated that

to

core,

they

indicate

a half.

the

because

was

that

i) all

do

that

(A)

is

the

either

suggests

accessible

peptidic

that

by

time

hydrogens

in

not

which

the this

means

repressor

determining words

hydrogens

unaffected

repressor

/ 7

exchangeable

SO slowly

of interaction.

must

fact

found

of

they

of -lac

result

are

of the

labile

of new

are

curves

sample

some

this

rates

or

of number

repressor

to the

plays

same

lac

; ii)

a half

per protomer as a function of the complex IPTG-lac repressor : 0.2 M potassiumphosphate, Incubation time 16 hours and

Buffer (DTE).

I 5

(hi.1

proton (0),

properties)

acids

of the

the

TIME I 6

L 4

disappearance

kinetic

of

number

about

of

RESEARCH COMMUNICATIONS

! 3

(A).

the

same

hydrogens

their

gled

example

appearance

exactly

belled

bute

(for

AND BIOPHYSICAL

to

and are

and

its

to be

measured

of the

headpieces

not

core

tan-

peptidic by

our do

not

Vol. 88, No. 1, 1979

appear the

in

the

BIOCHEMICAL

HX

absence

of

curve

of

effect

of the

Techniques absorption to

the

-lac

veals ve

did

not

of

the

upwards. sor

presence

vents

or

only

blocks

represent

i) we tal

do

not

or

ever hydrogen

fact

ne

repressor

the

the

not

involved

pieces” iii)

are IPTG the

levant

to

and

a large

in

good

our

contact induces

allosteric

the

in with

the

structuration

solvent

inducting

a central

Such (3).

and

the

of

some

to

IPTG

the

core chanreor-

of the

whole

hydrogen

interpretation

is

selectivity

in

li-

of pro-

60 suggests

that

this

core. “headpieces”

core

hydrogen

of the last

bloWhat-

upon

internal

: i) the

fast

to

atoms.

binds

of the

change

effect

due

a structural

and

can

experimen-

from

50

This

our

high

pre-

because

conformational

an

repres-

number

this

The

that

-lac

of IPTG

change

allowing

role.

the

blocking

IPTG

of new

“headpiece” suggests

curve

repressor

either

residues

a conformational play

&

in

core.

study

HX

entirely

the

when

involved

Weber

acid

between

extent

place

the

the

of hydrogen

the

formation

and

amino

be

a conformational

the

shifts

This

attribute of

result

from

of Geisler

“headpieces” the

may

or

conclusion

binding

which

atoms

cur-

under

release to

are

HX

binding

labelled

1 re-

the

hydrogens

might

and

of figure

with

the

of IPTG

change

exchanging.

fully

by

difference

binding

data

not

of blocked

takes

“headpieces”

to

that

change

blocking

between

In

is

reasonable

involving

a hinge

obtained

difference

“headpieces”

conclusion

forms

this

is

headpieces

cleavage

region

it

the

curves

is

of blocking

no

repressor

number

repressor

The

intact

from

or

the

does

atoms

; ii)

molecule

between

teolytic

-lac

dichroism

(12,13).

seen

for

structure.

of IPTG the

it

of the

hydrogen

of the

with

the

that

blocked

to

a conformational

that

ganization

bonds

limit

difference

The

binding

accounted

comparing

core

of IPTG

incubation

to

tryptic

10 hydrogen

if

atoms

The

-lac

absence

readily

circular

parallel

a superposition

demonstrates ge.

and

of

exact

binding.

essentially

be

core

when

binding

the

know

to

the

its

a lower

conditions

cking

its : the

about

on the

differences

to

whereas

can

fluorescence,

any

and

From

in

show

repressor

headpieces

difference

core

&

like

repressor

a marked

the

AND BIOPHYSICAL RESEARCH COMMJNICATIONS

lac

conclusion

: ii)

are the

“head-

exchange

;

repressor may

in be

re-

of IPTG.

References (1) (2) (3) (4)

Bourgeois, S. and Pfahl, M. (1976) Adv. Prot. Chem., Muller-Hill, B. (1975) Prog. Biophys. Molec. Biol., Geisler, N. and Weber, K. (1978) FEBS Letters, 87, Platt, T., Files, J.G. and Weber, K. (1973) J. Biol.

128

30, l-99 30, 227-252 215-218 Chem., 248,

llO-

121

Vol. 88, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(5) Jovin, (6) Laiken,

J. M., Geisler, N. and Weber, K. (1977) Nature, 269, 668-672 S. L., Gross, C. A. and Von Hippel, P. H. (1972) J. Mol. Biol., 66, 143-155 (7) Ohshima, Y., Matsuura, M. and Horiuchi, T. (1972) Biochem. Biophys. Res. Commun., 41, 1444-1450 (8) Matthews, K. S., Matthews, H.R., Thielmann, H. W. and Jardetzky, 0, (1973) Biochim. Biophys. Acta, 285, 159-165 (9)Brochon, J.C., Wahl, P., Charlier, M., Maurizot, J. C. and HBlBne, C. (1977) Biochem. Biophys. Res. Commun., 3, 1261-1271 (lO)Englander, S. W., Downer, N. W. and Teitelbaum, H. (1972) Ann. Rev. Biochemistry, 41, 903 -924 (11) Englander, S. W. and Staley, R. (1969) J. Mol. Biol., 3, 277-285 (12) Matthews, (13) Maurizot, (14) Muller-Hill,

(15) Geisler, (16) Englander,

K. S., (1974) Biochim. Biophys. Acta, 359, 334-340 J. C. and Charlier, M. , unpublished results B., Beyreuther, K. T. and Gilbert, W. (1974) in “Methods in Enzymology”, XXI, part D, L. Grossman and K. Moldave eds, pp. 483-487, Acad. Press. N. and Weber, K., (1977) Biochemistry, 16, 938-943 S. W. and Englander, J. J. (1972) in “Methods in Enzymolopart C., C. H. W. Hirs and S. N. Timasheff eds, gy”, XXVI, pp. 406-413, Acad. Press.

129