Time resolved spectroscopy of the tryptophyl fluorescence of the E. coli lac repressor

Vol. 79, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TINE RESOLVED SPECTROSCOPY OF THE TRYPTOPHYL FLUORESCENCE OF THE E. COIL

J.C.

Brochon.

Ph. Wahl,

REPRESSOR

LAC

M. Charlier.

J.C.

Naurizot

and C. Helene Centre

de Biophysique

Moleculaire,

45045 Orleans

September

29,

Cedex,

C.N.R.S.,

France.

1977

Summary The tryptophyl emission of the lac repressor of E.coki has been studied by pulse fluorimetry, using the monophoton sampling technique. The fluorescence decay has been found to vary with the emission wavelength, which was satisfactorily accounted for, by considering that the total emission was the superposition of two components having different spectra and decay times (Almax = 322 nm, ~1 = 3.8 ns ; ALmax = 344 nm, TV = 9.6 nsl. From a comparison of these results with studies of mutant repressors having one tryptophyl residue only ISomner et aZ., 19761 it is concluded that the emiters can be identified with residues 190 : number 1, and 209 : number 2. The interaction of the repressor with the inducer IPTG causes a decrease of the decay times, while spectrum 1 is red shifted and spectrum 2 is blue shifted. Residue 203 appears to be located close to the IPTG binding site. Introduction kc tophyl

repressor

residues

observed

that

phan emission, ding

from E.coZi

per

protomer,

is a tetrameric at positions

the fluorescence and that

of the

the

to the Stern-Volmer

quenching

equation.

one tryptophyl

residue

fluoresced,

and quencher

accessibility.

[IPTGj

containing

190 and 209.

protein

was

by iodide These

residues

showed that to a blue

of E.coZi,

Sommer

could

concluded

both

led

Laiken

of trypto-

be analyzed

accor-

that

only

either

had the same emission

binding shift

two tryp-

et aZ.('l9721

characteristic

ions

authors

or that

They also

propyl-6-D-thiogalactoside

protein

of the inducer

iso-

of the fluorescence

spectrum. Using

mutant

strains

repressor

molecules

tyr_osine.

The fluorescence

Copyright All rights

0 1977 by Academic of reproducfion in any

where

one of the

Press. Inc. form reserved.

quantum

et

two tryptophyl

yields

of these

aZ.

(19761 residues

obtained

Zac

was replaced

two molecules

were

by

identi-

1261 ISSN 0006-291X

Vol. 79, No. 4, 1977

cal.

BIOCHEMICAL

But the fluorescence

placed

by a Tyrl

A 209 [where relative ding

Trp

to the led

was shifted 209 is

properties

the

effect

of the time

inhibited

inducer

In order

to obtain

two tryptophyl

resolved

properties on their

spectrum the

study

wavelength,

of the

spectra Materials

[Donzel

method.

the

direction. on %ac repressor quenching

the

of the

residues

was

same kinetics

of only

on the type

as

one tryptophan

com-

excited decay

is

state

This

(cycloalanyltryptophan

spectroscopic

lifetime1

measured

depend

as a function to resolve

method

1972 ; Brochon

characterized

the

in sane cases

components.

properties

we have used the

known that

(spectrum.

and Auchet.

fluorescence

repressor,

is well

may be possible

in several [Wahl

It

fluorescence

conformations et a%.,

opposite

plots

and IPTG bin-

two tryptophyl with

information in the wild

cyclodipeptides

had two different

slopes,

Complete

photodegradation

direct

it

protein

of proteins

to show that

decreased

residues If

1977).

to repressor

Stern-Volmer

modifications

activity the

in

190 is re-

binding.

spectroscopy

environment.

the emission

et al.,

residues

of tryptophyl

spectra

one of the

that

the

had different

when only

IPTG binding

fluorescence,indicating pletely

ions

Trp

as compared

Mclreover,

of photochemical

(Charlier

was observed

photodegraded.

by a Tyrl.

by iodide

A 190 (where

wavelength

of the fluorescence

was studied

fluorescence

of repressor

to shorter

replaced

quenching

to a shift

Recently,

spectrum

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of

the total

has been used in

et al.,

19741

and also

and cycloglycyltryptophanl by two different

fluorescence

19741.

and methods

E.coki lac repressor from strain BMH 493 [a gift from Prof. 8. MiillerHill1 was purified according to Killer-Hill et a%. (19741. The purity of the protein was checked by polyacrylamide gel electrophoresis in the presence of Concentrations were determined using an extinction SDS and mercaptoethanol. coefficient cz8s = 21400 per protomer (Charlier et a%., 19771. The buffer used in this study was 0.2 M potassium phosphate and 0.1 mM dithioerythritol. Isopropyl-B-O-thiogalactoside (IPTG) from Sigma and used without further purification. 10w3 M solutions not show any fluorescence nor absorbance in the 250-300 nm range.

pH 7.2. was obtained of IPTG did

Fluorescence spectra were recorded with a Jobin-Yvon speetrofluorimeter. The spectra were corrected for photomultiplier tube sensitivity and monochromator dispersion and transmission.

1262

Vol. 79, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pulse fluorimetry studies : Single photon counting pulse fluorimetry studies were oerformed with an apparatus previouslv described [Wahl et a%., 19741. Excitation at 296 nm Ibanbwith 10 nml was provided by a hydrogen flash lamp built in our laboratory. Excitation and emission wavelength were selected by Elaush and Lomb monochromators. The photomultiplier was a R.C. A 8850. The amplitude selection of anode pulses allowed to use the fast counting technique. The parameters of the fluorescence into account the response of the apparatus. measuring the transient fluorescence of a (decay constant = 0.96 nsl IWahl et a%., obtained by computer programs and checked ted residual R and the deviation function

decays have been obtained taking This response is evaluated by standard solution of paraterphenyl 19741. The decay parameters were after examination of the mean weigh(Brochon et al., 19761.

Principle of time resolved spectroscopy [Wahl et a%., 19741 : Suppose that the protein contains two fluorophores, having monoexponential fluorescence decays, and emitting independently, i.e. no-transfer does occur from one to the other. In the most general case, the two spectra are different, but overlap. Let us call tl and TV the time constants of the exponential decays. The measured fluorescence decay at wavelength A can be written as : I[X,tl

= Cl(h)

exp[-t/r,1

Using a spectrofluorimeter total fluorescence intensity the two fluorophores F4lXl F(X)

= F,(A)

F,,[Al and F2[X1 follows :

*

c

1 IX1

exciting lamp, one measures the sum of the contributions

T

the of

(21

F201

may be expressed

=

[II

exp[-t/Tfl

with a continuous which is F(hl, and F2Ihl

CqCAl F1(i)

+ C2(Al

as a function

of the

decay

parameters

as

Tl

1 + C2(XJ

T’2

F(A) [31

C2(Al F2cA1

=

T2

c IX1 T 1 + c$h) 1

T2

F(X)

The ratio S /S of the areas under the two spectra F,,(A) and F2[Xl gives the ratio of the gontribution of each species 1 or 2 to the total fluorescence. The quantity

may be expressed species : c

=

!kl KF2

in terms

x

El -x f2

of the concentrations PJ iA23

[Alland

[A21 of the

two emiting

I51

where Ed, E.g a;d KF~B KF2 are respectively the molar absorption coefficients at the excl atlon wavelength, and the radiative deactivation rate constants the two fluorophores.

1263

of

Vol. 79, No. 4, 1977

In CAlI

our

BIOCHEMICAL

case, the Consequently KF, E1

= CA-L]. c=

K

protein :

contains

two

tryptophan

residues,

and

7

we

have

(6) 2

F2

If the absorption spectra of the they are shifted one with respect cl/c2 may be different from unity, From

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

two

tryptophans

to the other particularly

by at

are different, for a few nanometers, long

example if the ratio

wavelengths.

view, the case where the emitters energy from meto the other, is identical to two conformations of the same molecular spe-

the

emission kinetics transfer part of their excitation the case where an exchange between cies occurs in the excited state. the spectra determined by equation of the two species IDonzel et al., (61 are no longer valid.

point

of

Under these assumptions, it [31 are linear combinations 19741. But in this case,

can

be

shown

of the relations

that

spectra (5)

and

Results Figure

repressor to

nm,

to

became

faster

ves

the

by

decays, The

with

time

amplitudes

C, 1.

of the the

cays.

The

and

CZ depend observe

= 0.6 R

these

decay

ns

on

the

and

~~2

of

best

= 4.2

number

fluorescence

fluorescence two spectra

spectrum

decay

is

ns.

well

corresponding

was

two

into

but

type

described

the

very

of

R

a sum

to these

two decays

(maximum

wavelength,

half

equation

(41)

of

are

are

slightly

is

not

the

mean

of We

not

9.6

exponential

two

inaccurate. did

and

Results

determination

components. by

3.6

calculations,

components its

to

1 decay

a sum

cur-

exponential

residual

these

equal decay

two

equal

the by

By

reduced,

1 in

of

weighted that

chosen

transient

wavelength.

fitted

terms

exponential

decay

mean

M lac

fluorescence these

respectively

suggests

was

The of

emission

the

was considerably two

= 10 nml.

-5

3x10

was

a superposition

were

This

of

wavelength

Analysis

r2

that

wavelength. This

decomposition

that

and

(Ah

yielded

‘I~

We can

~~~

of

method

intensities

excitation

wavelengths.

constants

residual

weighted

lution

emission

exponential.

decays,

The absorption

mentioned

at short

strictly

1

short

above

increasing

the

at

table

fluorescence

transient

tyrosine

amplitudes in

the

concentration).

avoid

whose

given

shows

(protomer

296

ns.

IA

verified

that

the

reso-

modify

Thus,

we can

two

exponential

are given

the

assume de-

on figure

8. Spectral

and

C

parameters

parameters defined

by

1264

are

given

height in

table

width,

2.

area

ratio

Vol. 79, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

60-

46-

26-

10 S)

A

Fig.

1 -

A. Transient 4 different tion (curves

300

0

fluorescence of the lac repressor emission wavelengths. g[t) is the : A, 320 nm ; B, 340 nm ; C, 360

8. Resolution of the %ac repressor into two components characterized [curve 11 and ~2 = 9.6 ns (curve

Transient

[saturating

fluorescence concentration1

curves

of

In

same

manner

sient

curves

in

two

tively

equal

to

3 ns

the

repressor

emission and shown

on

cal

or

was

recorded

The

excitation

ratio

F,(X)

figure

In

obtain

as

order

and

7.6

in the

presence

in

the

presence

and

absence

of

inducer,

we could

ns, II.

and

2).

studied.

decays, and The

F (A1 2

at 296 nm for response func. 390 nm).

spectrum times t

also

exponential

to determine absorption

for

the

with

spectral

time

from

the

and

the

‘Tom3

the

IPTG.

tran-

the

and

TV respec-

C2 depending

on

are given kinetic

decay of

decompose r1

31 ns

M IPTG

absence

constants C,

(curve = 3.8

1

2A shows

in

parameters

determined

of

Figure

amplitudes

acceptable

whether spectra,

different

bandwith

between

the

fluorescence decay

456

the

in table

measurements

2 are

28.

different

an

in

(table

spectra

been

fluorescence

wavelength

the

has

excited apparatus nm : 0,

by the

repressor

of

400

350 x.m

was

error

the fluorescence

emission chosen on

the

the

two

tryptophyl

the

excitation

spectrum

wavelengths,

namely

as narrow measured

intensities

as possible parameters. at

1265

residues

320

and

have

of %ac repressor 320

and

(3 nml Figure 365

identi-

nm.

in

365

nm.

order

to

3 shows as

the

a function

of

Vol. 79, No. 4, 1977

Table

I

-

Decay

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH

in

sidual

parameters of tryptophyl emission wavelengths R is defined b the

R=

i!n

where

rent

'I",;

and

decays absence

in

obt,3ined of IIWI.

at

diffeThe re-

relation

i2

k!,

fluorescence presence

COMMVNICATIOYS

I')

I Lx

and

1:

are

the

counts

il-1

channel

E?X

k for convolution

the

em

=

3.8

- IPTG ns f,

315 320 330 340 350 360 370 380

0.72 0.64 0.48 0.35 0.28 0.18 0.08

n

is

and for number.

fluorescence the channel

the

calcclated

‘1

+ IPTG L

= 9.8

ns

L

c1

nm

transient and

respectively,

'1

i

experimental

R

0.28 0.36 0.52 0.65 0.72

3

ns

=

T2

7.6

ns

P

-,i

c1

1.8 I.8 1.4 1.3 1 .Ol 1.4 0.9 1.2

0.82 0.92 1

=

T1

0.34 0.34 0.32 0.24 0.21 0.13 0.12 0.09

-1

0.66 0.66 0.68 0.76 0.79 0.87 0.88 0.91

1 ,, 1 2 6.7 1.75 1.9 1.9 1.5 1.4

---I

the

excitation

wavelength.

excitation

wavelength

measurements

up

very

were

ratio

This to

300

did

nm.

inaccurate

Above due

to

not

change

300

nm.

the

low

appreciably

with

ratio

this

the

decreased,

fluorescence

but

intensities.

Discussion

In sor

may

~~

and

to

each

the

be ~~ time

maximum

respectively. two

tryptophan

the

considered do

as

not

the

are

22

nm most

residues We

two

on

constant

decay,constants. of

the

depend

The

tryptophyl

emission

tryptophan

sum the

are

shifted

and

6

of

see

the

two

emission

nm

in

the

exponential

decays.

wavelength.

The

absence

below

209

have how

residues.

1266

each in of

specific to

to and

interpretation

and

decay

fluorescence

respect

with

reasonable 190

will

band,

identify

of

The

time

spectra other.

repres-

%ac

constants

corresponding The

shifts

at

the

presence

of

IPTG

these

results

is

that

fluorescence the

two

spectra

spectra

the and

with

those

Vol. 79, No. 4, 1977

Table

II

BIOCHEMICAL

- Parameters repressor h max

of the two fluorescence tryptophyl residues.

nm

Ah'* nm

'I,, ns

-1PTG

340

57

3.8

+IPTG

331

50

3.0

'1 max nm

The contribution mined the

from absence

rate

it

‘f

344

55

0.21

0.54

327

40

7.6

333

50

0.13

0.33

1 to the

represents

respectively.

is

C

9.8

total

may be deter-

(61,

if

at the excitation C parameters

which

not realized,

fluorescence

to equation

absorptivities the

2

18 % and 12 % in the presence

According

the two tryptophans,

2 shows that

'l/S

AA21R nm

%ac

42

and the molar

for

by the

322

It

'2 max nm

emitted

;$

S1/S2.

of IPTG,

components

Ax? nm

of spectrum

ratio

constants

identical ble

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

radiative

wavelength

should

means that

the

and in

be equal

these

were

to 1. Ta-

conditions

are not

fulfilled. If

E~/E~ was not equal

due to a shift

of one of the absorption

shape and extinction between

the

constant. from

I) indicated found

the ratio

Tryptophan

conditions

by its in proteins

may lead

only

of

300 nm, the

ratio

of formula

practically different

(5)

may not

: probably

et a%.,

of tryptophan part

of the emission

1267

in

several

Such a situation

one tryptophyl

no 1 may transfer

no 2. The overlap

be

to a variation

not be very

the validity

decay.

1972 ; Brochon quenching

below

should

cl/s2

for

non monoexponential

to the

than

most probably

at 320 nm and 365 nm is

no 1 exists

containing

rather

3 shows that

may occur

residue

would

range.

Two possibilities

21 The residue residue

that

the

1971 ; Wahl et Auchet, tions

monitored

wavelength

Nevertheless, be fulfilled.

spectra

indicates

in this

spectra

Figure

coefficients.

excitation This

unity

at 296 nm, this

to unity

residue 19761.

environments, has already

(De Lauder

Some of these

as been

et a%., conforma-

1. of its

excitation

and absorption

spectra

energy is

on the favourable

Vol.

79,

No.

4,

1977

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

I lo5 A B C

10

It 60-

Id IF?%

BO-

40-

20DV; +lO -

: .‘---s.j, *\

10s

300

350

B

hnm

I 450

400

c

C A

Fig.

2 - A.

Influence

upper

of

curves

wavelength wavelength of IPTG. by

IPTG

on

the repressor transient fluorescence. The the transient fluorescence at an emission nm (curve Al in absence of IPTG and at emission nm in absence (curve B1 and in presence (curve curves represent the deviation functions defined

represent of of

390 330

The

lower

&

=

Ik

c

J k

- Ik

C)

ex

Ik ex

k

where I and A, 8, C’refer

I have the same meanings as in the legend of Table I. tExthe upper transient fluorescence. For the deviation function A, the convolution has been calculated with a single exponential function of time constant 9.6 ns. For El and C the dashed curves correspond to convolutions calculated with the best single exponential functions ; the continuous curves correspond to convolutions calculated with the best sum of two exponential functions the parameters of which are given in Table I.

8. Resolution of the presence of IPTG (curve decay time ~1 = 3 ns

to a transfer

from

1 to

In

enough

to

explain

above,

in

the case of transfer,

spectra makes

the

2.

of

the

emiting

them

not

very

observed

species. different

lac

repressor

fluorescence into two components 11 and ‘cz = 7.6

31

[curve this

a transfer

case,

C value

in

the

spectra

But

the

from

the

the true

1268

efficiency

absence F,,(X)

small

spectrum

value

spectra.

emitted

characterized ns (curve 21.

of

and

of IPTG.

F2(AI

of the Occurence

in the

by

17 %

is

As mentioned

are

not the

transfer of

true

efficiency transfer

Vol. 79, No. 4, 1977

BIOCHEMICAL

200

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

300 “Ill

290

Aexc Fig.

3 - Ratio of the fluorescence and 365 nm as a function

implies

a relative

proximity

in good agreement

with

(1977)

the

to explain Our results

to either sults emission it

is

those

photochemical

It

spectrum

repressor,

should

Moreover,

be noted

emissions

repressors prevent

in the

directly

It

is

the

interesting

et a%.

data

1 could

with

1 and 2 spectra to compare

our

For the A 209 mutant,

those

for

re-

the

the A 190 mutant',

of table

be attributed

that

the difference

is

13 nm, whereas

it

quantum

of the

study,

we find

1 and 2. The replacement

of course served

In our

be

2 suggests

to tryptophan

that

190 and

209.

the fluorescence

to each other.

would

by Charlier

190 is at 325 nm, whereas of these

of A 190 and A 209 mutants

mutant

us to attribute

by Sonmer et a%. (19761.

at 336 nm. Comparison

2 to tryptophan

proposed

This

at 320

experiments.

residues.

maximum of tryptophan

spectrum

residues.

one of the interpretation

obtained

type

emitted by %ac repressor wavelength.

of the two tryptophyl

do not allow

in wild

2.

intensities of excitation

190 or 209 tryptophyl

with

310

might

modify

yields

the maxima of the

is

our

an important

. This

two studies.

1269

22 nm for

two mutants

difference

of one tryptophan

the environment

Trp + Trp transfer

between

by a tyrosine

explain

spectra

1 and

are very

close

in the yields

of the remaining could

spectra

of

in the tryptophan

the differences

and ob-

Vol. 79, No. 4, 1977

Upon

IPTG

repressor

the

species

are

-free

In

and

whereas

for

with

‘12 observed

in

radiative

of

8 nm,

and

were

we observed

A 209

and

presence

of

rates

2 and

[Table spectrum

5 nm.

K

attributed

7.6

nsl.

Such

the

209

tryptophan

of

Charlier

residue

tryptophan

a change

could

a%.

IPTG

in

(19771 in

209,

be

residue

et

and

to

the

the

increase

decrease

The

is

constants

the

shift

were

This

by

results

18 and

results

decay

two

a blue

respectively.

explained

the

These

2 exhibits

the

of

of

Figures

Similar

of

and

fluorescence yields

here

Fl constant,

quantum identical.

be K

the

practically

change

could

that

the

mutants

The

constants

that

by

A 190

IPTG

showed

that

redshifted

assignment.

deactivation

(19721

presented

1 is

spectral the

ai?.

by

those

spectrum

of our

et

of IPTG,

spectra

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

repressor-

liganded

presence

the

in favor

Laiken shifted

agreement

of 11 nm. tained

binding,

was blue

in good

281.

BIOCHEMICAL

obwell

T,

and

of

the

of the

~2

F2’

is

rather

result

of

important

a close

the

complex.

This

concerning

the

proximity

[from

contact

least

to

IPTG

supports at

ns

between

conclusion of

9.8

the

one

and

results

tryptophyl

complex.

Conclusion

of

the

The

use

Zac

repressor

- The cence

of

The

protected

the

conclude

residues

decay

maximum

residue

of

constants.

More

209

fluorescence at

against

- The of

to

in that

the

study

of

the

fluorescence

:

each

protomer

have

than

80

% of

of

residue

is

therefore

the

different emission

fluoresis

from

209. -

and

spectroscopy

us

tryptophyl

and

tryptophan

resolved

time

leads

two

spectra

that

of

tryptophans,

The

method

nm.

solvent

inducer

two

344

wavelength

IPTG

The

first

one

190

is

at

322

probably

more

nm and burried

effects. induces

different

and

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