Vol. 74, No. 2, 1977
BIOCHEMICAL
PHOTOCHEMICAL
REACTIONS
EFFECTS
Michel
CHARLIER,
Centre
de
Received
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ON
INDUCER
FranGoise and Claude
Biophysique
December
OF
--LAC
REPRESSOR.
BINDING.
CULARD, HELENE
Jean-Claude
Molkculaire,
45045
MAURIZOT
Orleans
Cedex,
France
7,1976
Summary Upon U. V. irradiation, lac repressor exhibits of its fluorescence intensity. Completequenching is observed one of the two tryptophyl residues is modified per protomer. ding activity decreases with the same kinetics as fluorescence, is due to the total inactivation of photodamaged protomers. that one tryptophyl residue, the one which is photochemically is involved in the IPTG binding process.
a decrease when only IPTG binand this It is proposed modified,
Introduction The suitable
control
biological
of the
lac of
dies
(see
large
system
results
have
review
by
amounts
effecters
of -lac
Lac
(isopropyl
Under
trum
is
sible
role
obtain modified.
been
from ref.
of
and
are
Using
studied
repressor
replaced
by
tyrosine.
in
this
binding whose
approach,
molecules We
with have
Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
been to
shift
(8) .
In
the
known,
of
and
to
two modify
stuobtain
number
(6,7)
190
the
inducer
repressor. 209
- per
fluorescence
was
tryptophyl the
the desirable
replaced
coworkers
(9,
repressor
posto
or
residues lac
pro-
spec-
investigate
is
of
.
of
and
it
SOMMER
a great
to
&
to
ring(s)
attempted
possible
its
process,
of the
sequences
biochemical
irradiated -
a very
and
binding
order
one
is
The
studied
indole
690
coli
of a large
residues
IPTG
E.
and
effects
related
a blue
molecules
are
now
to U. V.
binding
a genetic
It is
the
tryptophyl
tryptophan(s)
repressor
in
genetic
have
conditions, upon
(3)
4).
anti-inducers)
experiments
native
observed
-lac
obtained
(5)
two
operon
studies.
operator
repressor
contains
-lac
of -lac
p-D-thiogalactoside)
repressor
tomer.
the
physico-chemical
and
and Our
of
MBller-Hill,
(inducers
IPTG
for
(1,2)
repressor
number
system
10)
Vol. 74, No. 2, 1977
by
U.
V.
irradiation.
has
the
highest
ted
a priori
tryptophan when
cross that
The
does
Materials
and
250
are
nm
effects
variations
in
is
group,
could
be
the
the
used
S-S
with
IPTG tryptophan
will
be
reactive
.
of irradiation and
expec-
protein
most
(11)
which
of -lac binding,
repres-
and
we
modification.
Methods
IPTG p4d
It
residues
dichroi’sm
their
E. coli lac Dr. B. MULLER-HILL) coworkers(2). C oncentrations absorption measurements, This value was obtained good agreement with that of the protein.
duct.
the
circular
any
damage
than
studied
correlate
contain
photodegradation.
these
longer
have
not
photochemical
of wavelength
to
for
since
on fluorescence, tried
protein
major
degradation, light
have
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
section
the
We sor
BIOCHEMICAL
IPTG
was
repressor from strain BMH 593 (a gift from was purified according to MULLER-HILL and of lac repressor were determined from assumyg that %2gO = 21400 per protomer. by microkjehldahl nitrogen analysis, in very calculated from the amino acid composition
(isopropyl purchased
P-D-thiogalactoside) from CEA,
was
a Sigma
pro-
Saclay.
Irradiation experiments were made in a buffer containing 0.2 M potassium phosphate, pH 7.2, and 0. 1 mM DTE (dithioerythritol). To avoid aggregate formation occuring upon irradiation, it was necessary to use protein concentrations smaller than 5~10~~ protomer.
M
IPTG activity was measured by equilibrium dialysis, according to GILBERT and MULLER-HILL(13). TMS II buffer was used : 0.2 M K Cl, 0. 01 M Mg acetate, 0. 04 M tris/HCl pH 7. 5, 0. 1 mM EDTA and 0. 1 M mercapthoethanol. p4C] IPTG was lo-’ M, giving 500 cpm per 100 ml. Three separated samples were dialyzed for each irradiated solution, the dispersion of the results being less than 5 ‘$. Under these conditions, the measured activity is directly where K is the equilibrium constant of the reaction proportional to Kxn, IPTG and n the determined
OSRAM a band in the a KIPP
number from
t Rep
of IPTG equilibrium
1
Complex
binding sites per dialysis data
Irradiatiomof lac HBO 200 watts mercury between 250 and 400 nm. wavelength range transmitted and ZONEN thermopile.
protomer. K and in the irradiation
n were buffer.
repressor were performed with an lamp. A filter MT0 H 325 a isolated Total incident light energy fluence by the filter was measured with
Fluorescence spectra were recorded with a JOBINYVON spectrofluorimeter modified to correct for fluctuations in lamp intensity(14). Th e a b sorbance of the samples at the exciting wavelength was smaller than 0. 05 to keep the fluorescence intensity proportional to the concentration of fluorescent species.
Vol. 74, No. 2, 1977
Figure upper lower
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
1 : curve curve
A. Relative variation with incident light energy fluence, of -a-*: circular dichroism intensity at 222 nm (A’/&.io) : fluorescence intensity at 340 nm (A,, = 280 nm) binding activity AI/AI, ( -0 -0 - ) for IF/IF ( -D-W) and IPTG irradiated alone. -lac repressor middle curve : fluorescence intensity at 340 nm (hex = 280 nm) IF/IF ( -a - 0 -) and IPTG binding activity AI/AI (-++-) for & repressor irradiated in the presence of o1O-3 M IPTG. I3.
Correlation
IF
curve
- IF
=f(
AI
IF 0 tive fluorescence full circles : lac repressor irradiated
a ROUSSEL-
decrease repressor in
Circular JOUAN
the
and relative irracliated presence
dichroism II apparatus.
;
-
A I)
between
rela-
IO
loss of IPTG binding alone - open circles of 10m3 M IPTG.
spectra
were
recorded
activity. : -lac
at
2°C
with
Tryptophan concentrations were determined according to a procedure derived from SASAKI(l5). The protein was first digested at pH 8 during 5 hours at 3 1 “C, with trypsin and A-chymotrypsin, both at a final concentration of 0. 003 % (w/v). A second proteolytic cleavage with pronase (0.003 % w/v) was performed during 7 hours Guanidinium chloride was used as denatuunder the same conditions. ring agent, at a final concentration of 5 M. After 30 minutes, the fluorescence spectra were recorded. Above 340 nm, where only tryptophan emits, the fluorescence spectrum of the digested protein is identical to that of free tryptophan. Assays on intact lac repressor gave a number of tryptophan residues per protomer equalto 2 f 0. 1, demonstrating that the procedure is quite suitable for this protein.
Results Upon repressor
exhibited
U. V. a rapid
irradiation
(250
decrease
of
692
nm its
< h C 390
fluores.cence
nm), intensity
-lac
:
Vol. 74, No. 2, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
SF CM-‘) IO5
0
’
r
1
0.5
Figure 2 : Scatchard plots for the binding of IPTG to -lac repress0r.r is the fraction of liganded protomers, and Cf is the concentration of free IPTG. The inserted table gives the binding parameters compared with the decrease of fluorescence intensity. A, unirradiated lac repressor, B, irradiated with 2. 1 x lo5 Jmm2 (total incidentlight) C, irradiated with 4 x lo5 Jmm2.
(fig,
1A).
when
excitation
For
The
was
fluorescence was
of photolysis
3~10~
rescence)
of the
wavelength
durations
about
shape
Joules/m’
chosen
longer
blue
when
shift
the
at
than
of incident
a small
observed
spectrum wavelength
and
40
maximum
fluorescence
not
than
295
(corresponding %
of loss
wavelength
was
modified,
longer
10 minutes
light,
of the
was
excited
at
290
to
of of and
nm.
fluo2 and 280
4 nm nm
respectively. Long broad and
and an
band 345
weak
irradiation
band,
spectrum
interfered
(30
with
the
minutes)
a maximum
centered
with
at 345
around
nm.
fluorescence
led
to a new 435-440
Nevertheless,
intensity
nm, this
measurements
at
nm. IPTG
creased
upon
intensity
and
figure of
of
emission
excitation
never
times
8 nm
IB.
binding
irradiation
The
of the
that
of
activity IA).
(fig. the
binding fluorescence
IPTG of
of The
activity
IPTG
to
spectrum
the
decrease are
lac
78)
-lac
linearly
repressor . Similar
repressor of
(Kxn) the
effects
de-
fluorescence
related, leads
also
as to a blue were
shown shift obser-
on
BIOCHEMICAL
Vol. 74, No. 2, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
II ,Fo_ /'
1'0
0.75-
/
T,-T TO
l -•-
I 10
S Incident
light
I 15
energy
flucnce
-3Lx1O5
JxM
f$fg
: Relative variation of the decrease of the fluorescence : fluorescence intensity) and the number of destroyed content) with incident light energy fluence. (T : tryptophan upper part shows the ratio between these two parameters.
phan The
ved
with
irradiated
diation
(and
-lac
therefore
of
In both
IPTG
times
binding
slower
the
same
as
of
bound
the
intensity
No
occurs
change
disappears. markedly
band
indicates
against
photochemical
‘$
the
(fig.
that
the
lA),
two
irra-
IPTG, 5
which
indicates
damages.
parameters
remains
1B).
relative
variation
characteristic of the
of decreased
(fig.
the
of
of
fluorescence conformation
of the the
peptidic
(or
IPTG
of
the
circular bonds.
activity) protein
is
not
affected.
irradiated
straight
75
intensity
repressor
IPTG
of the
Quantitative to
of shows
duration
concentrations
between
1A
until
This
saturating
IPTG
absence
the loss).
fluorescence
correlation
Figure dichroism
of and
of
fluorescence
unliganded
linear in
initial
presence
for
effect the
the
activity
than
a protective However,
the
independent
repressor,
intentrypto-
lac parallel
analysis
repressor lines.
is Binding
of the
shown
on
parameters
694
binding
figure
process 2.
are
of
Scatchard listed
in
IPTG plots
figure
are 2.
BIOCHEMICAL
Vol. 74, No. 2, 1977
The ted
repressor
Figure diated the
protein, protein.
range
determination
was
3 shows
the
relative
and
the
ratio
is
and
the
-lac
at
shorter
the
repressor
310
diation
is
is
nm. due
of the
due
The to
CD
protein
fraction
of
rescence
is
one
the
irra-
two
in in
a large
destruction of
the
of tryptotal
fluo-
the
other
the
modified
tryptophyl of the
photoproduct, close
first
one
a large
the
be is
- always
the
The
of
put
residues
the
upon
absence
change fluorescence.
to
that
irraof varia-
tryptophan
forward
half
weakly
spectrum,
conformational
quenching can
in intensity
3).
of
emit
shoulder
(fig.
does
one
or
by
a local
to
the
indole
residue and
the
tryptophan
is
not
residue
other
tryptophyl
modified,
for
fluorescence
Tyrosines
fluorescence
that
residue
(ii)
each
the
the
explain
of the
that
relative
the fluo-
:
and
group
of
tryptophyl
protein.
- is
fluoresce
at all,
the
in
is
is
same
same
quenched
either
all
by
conformational
modified
in
neither
in
protomers energy
change
all native
and transfer
three
types
tryptophan binding
third
of
protomers
209
modified,
bringing
a quen-
probability
of
ring. (190
second
or one
modified,
for
experiments
hypothesis
209) no
has
longer
reasons
can
should and are
the to
an
equal
emits
fluorescence
identical
to
those
In
this
when given
(ii). The
IPTG
Methods.
of
to
nm,
a small
hypotheses
tryptophyl
fluorescence
produce
responsible
irradiated
in
the
residues.
excludes
in
the
equal
325-380
modification
spectrum
nor
being
irradia-
destroyed
decrease
tryptophyl
tryptophan
modified
protomers
(iii)
to
decrease
decrease
only
ching
the
range
and
Several
the
is that
as
wavelength
wavelengths,
around
(i)
intensity
values
demonstrating slow
and
of tryptophan
these
as
Materials
U. V.
Conclusion In
of the
amount
in
intensity.
Discussion
tion
in
of fluorescence
relative
twice
content
described
loss
time,
residues
rescence
as
between
of irradiation
tophyl
of tryptophan
performed
The
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
be
exist
: intact
protomers
with
in
disagreement
discarded, ones,
protomers
tryptophan with
case,
190 the
with modified.
existence
of
Vol. 74, No. 2, 1977
three
types
the
two
has
been
of
and
15 times linear
plots
presented
equal
to only
for
of
properties,
and
tryptophan
keep
their
binding
that
of
wild
the
of
linear
these
and
are
respect
1.
The
can
in
activity
for
for
A lo0
species
a value
thus
of
n
conclude
irradiated
solutions
to both
fluorescence
and
“dead”
with
to
protomers
should
Scatchard to
We
that residue
type
extrapolate
present
found
two
to
intensity.
protomers with
one
extrapolating
2 are
unaffected
binding
to
fluorescence
types
(10)
A mixture
plots
figure
relative
protomers
one,
A 209.
coworkers
where
equal
Scatchard
two
A 209,
constant
in
the
and
and
a tyrosine
a binding
non
190
by
lower
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
SOMMER
A
replaced
with
that
protomers.
repressors
IPTG
give
BIOCHEMICAL
respect
:
IPTG these
two
properties. It is The
appearance
upon
absorption
band
rescence
quenching
one
intact
between in
being
yet
not
close
available
possible
to
the
the
of SOMMER
taining
only
that of
the the
two wild
tyrosine with
repressors type.
and
not
obvious
of that
transfer of
of
tyrosine modify
the
that
two may the
the
wild
mutants. induce fluorescence
one
type
protein
-lac
to
adequate information
other
mutants
about is
the could
probably
change of
con-
tryptophyl
half
that
replaced
can
replacement
yield
the
observed
a yield
When
would
it
with
built
molecules
the
the
repressor,
authors
conformational quantum
No
a tryptophan
Furthermore, a local
have
with
repressor.
from
The
contradiction
These
these
residue.
assumption.
who
mutants,
from
that
an
fluo-
transfer
requires
of the
apparent (10)
turn
(ii).
that
require
residues.
this
fluorescent
these
in
two
tryptophans.
modified
energy
fluorescence
also
is
photochemically
present
by
it
in
in
coworkers
are
But
is
energy
tryptophan
which
and with
possible
3 would
support
(i) species
it to
structure
or
two
due
figure
of the
(i)
of the
is
to unity
tertiary
and
one
in
eliminate
makes
modified
proximity
Hypothesis results
other
hypotheses fluorescent
nm
protomer
equal
on
between
350
presented is
and
and
each to
probability
orientation
chcrse
of a new
300
results
transfer
to
irradiation
tryptophan
experimental
is
difficult
be
two
by
compared tryptophans
occur, not
a
are
and be
of which
the
the sum
tryptophan might residues.
Vol. 74, No. 2, 1977
BIOCHEMICAL
We vement
of
interact
tryptophan
prevent
does
not
a local
with this
due
destroyed
6, and
repressor
but
that
as
that
tryptophan induces
preventing
IPTG
of binding
another
this
tryptophan,
may
destruction
CD,
of
imply
that
decrease
degradation
rate
its
by the
invol-
modification is
observed
would
CD
been
acti-
amino
last
acid
residue
suggesting
the
is
a very
results
least
indirectly,
of the
strongly the
IPTG,
efficient
least
one
that
the
IPTG
to
tryptophyl of
binding
is
trypto-
of IPTG
of tryptophan.
tryptophan
binding
of
ab-
modification
and
one
differential
binding
modification
that
effector
the
photochemical
photochemical suggest
in
that
of at
the
to bind
(8) ,
fluorescence (17)
that
ability
rate
by
environment
show
the
these
the
results
abolishes
shown
measurements
modifies Our
modifies
photochemical
effector,
direct
process.
sorption(l
phan
the
the
Tryptophan
possibility
excluded
same
It has
residue.
its
not
assumption
the
photosensitized
be
on
process.
Another
photosensitized
an
with
binding and
with
cannot
the
Such
IPTG
change,
it
to
unambiguously
sugar,
directly
Also
residue.
-lac
the
conformational
is
the
interaction.
interact
binding.
conclude
in
directly
may
vity
cannot
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
involved,
All at
process.
Acknowledgements We J.
MILLER
published
for
would
many
like
to
fruitful
thank
Drs.
discussions,
K.
BEYREUTHER
and
communication
and of un-
results.
References (1)
BEYREUTHER,
K.,
ADLER, Proc. Nat. K., ADLER, KLEMM, A. and (1973)
(2)
BEYREUTHER,
(3)
GILBERT,W.
(4) (5)
MCLLER-HILL, MULLER-HILL,
(6) RIGGS, (7)
BARKLEY,
them., 2, and MAXAM, 3, 3581-3584
K., GEISSLER, N. and KLEMM, A. Acad. Sci. USA, 70, 3576-3580 K., FANNING, E., MURRAY, C., GEISSLER, N. (1975) Eur. J. Bio-
491-509 A.
(1973)
Proc.
Nat.
Acad.
Sci.
USA
B. (1975) Prog. Biophys. Mol. Biol. 30, 227-252 B. (1971) Angew. Chem. Int. Ed., lo, 160-171 NEWBY, R. F. and BOURGEOIS, S. (1970) J. Mol.
A. D., 5J, 303-314 M.D., RIGGS, (1975) Biochemistry,
A. D., 14,
697
JOBE, A. 1700-1712
and
BOURGEOIS,
Biol. S.
,
Vol. 74, No. 2, 1977
(8)
LAIKEN,
(9)
SOMMER,
(10)
SOMMER,
(11) (12)
(13) (14) (15) (16)
(17)
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
S. L., GROSS, C. A. and Biol., 66, 143-155 H. , LU, P. and MILLER, lJ, 302a
VON
HIPPEL, J. H.
(1975)
P.
(1972)
Biophys.
J.
Mol.
J.
,
H., LU, P. and MILLER, J. H. (1976) J. Biol. Chem., 251, 3774-3779 SMITH, K. C. and HANAWALT, P. C. (1969) in “Molecular Photobiology”, p. 87, Acad. Press N. Y. MULLER-HILL, B., BEYREUTHER, K. T. and GILBERT, W. (1974) in “Methods in Enzymology”, Vol. XXI, part D, p. 483-487, Acad. Press N. Y. GILBERT, W. and MWLLER-HILL, B. (1966) Proc. Nat. Acad. Sci. USA,%, 1891-1898 TOULME, J. J. and HELENE, C. (1975) Biochemistry, BRUN, F., 2, 558-563 SASAKI, T., ABRAMS, B. and HORECKER, B. L. (1975) Anal. Biochem., 65, 396-404 MATTHEWS, K.S., MATTHEWS, H.R., THIELMANN, H. W. and JARDETZKY, 0. (1973) Biochim. Biophys. Acta. , 285, 159-165 MAURIZOT,J. C. et., to be published.
698
BIOCHEMICAL
PHOTOCHEMICAL
REACTIONS
EFFECTS
Michel
CHARLIER,
Centre
de
Received
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ON
INDUCER
FranGoise and Claude
Biophysique
December
OF
--LAC
REPRESSOR.
BINDING.
CULARD, HELENE
Jean-Claude
Molkculaire,
45045
MAURIZOT
Orleans
Cedex,
France
7,1976
Summary Upon U. V. irradiation, lac repressor exhibits of its fluorescence intensity. Completequenching is observed one of the two tryptophyl residues is modified per protomer. ding activity decreases with the same kinetics as fluorescence, is due to the total inactivation of photodamaged protomers. that one tryptophyl residue, the one which is photochemically is involved in the IPTG binding process.
a decrease when only IPTG binand this It is proposed modified,
Introduction The suitable
control
biological
of the
lac of
dies
(see
large
system
results
have
review
by
amounts
effecters
of -lac
Lac
(isopropyl
Under
trum
is
sible
role
obtain modified.
been
from ref.
of
and
are
Using
studied
repressor
replaced
by
tyrosine.
in
this
binding whose
approach,
molecules We
with have
Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
been to
shift
(8) .
In
the
known,
of
and
to
two modify
stuobtain
number
(6,7)
190
the
inducer
repressor. 209
- per
fluorescence
was
tryptophyl the
the desirable
replaced
coworkers
(9,
repressor
posto
or
residues lac
pro-
spec-
investigate
is
of
.
of
and
it
SOMMER
a great
to
&
to
ring(s)
attempted
possible
its
process,
of the
sequences
biochemical
irradiated -
a very
and
binding
order
one
is
The
studied
indole
690
coli
of a large
residues
IPTG
E.
and
effects
related
a blue
molecules
are
now
to U. V.
binding
a genetic
It is
the
tryptophyl
tryptophan(s)
repressor
in
genetic
have
conditions, upon
(3)
4).
anti-inducers)
experiments
native
observed
-lac
obtained
(5)
two
operon
studies.
operator
repressor
contains
-lac
of -lac
p-D-thiogalactoside)
repressor
tomer.
the
physico-chemical
and
and Our
of
MBller-Hill,
(inducers
IPTG
for
(1,2)
repressor
number
system
10)
Vol. 74, No. 2, 1977
by
U.
V.
irradiation.
has
the
highest
ted
a priori
tryptophan when
cross that
The
does
Materials
and
250
are
nm
effects
variations
in
is
group,
could
be
the
the
used
S-S
with
IPTG tryptophan
will
be
reactive
.
of irradiation and
expec-
protein
most
(11)
which
of -lac binding,
repres-
and
we
modification.
Methods
IPTG p4d
It
residues
dichroi’sm
their
E. coli lac Dr. B. MULLER-HILL) coworkers(2). C oncentrations absorption measurements, This value was obtained good agreement with that of the protein.
duct.
the
circular
any
damage
than
studied
correlate
contain
photodegradation.
these
longer
have
not
photochemical
of wavelength
to
for
since
on fluorescence, tried
protein
major
degradation, light
have
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
section
the
We sor
BIOCHEMICAL
IPTG
was
repressor from strain BMH 593 (a gift from was purified according to MULLER-HILL and of lac repressor were determined from assumyg that %2gO = 21400 per protomer. by microkjehldahl nitrogen analysis, in very calculated from the amino acid composition
(isopropyl purchased
P-D-thiogalactoside) from CEA,
was
a Sigma
pro-
Saclay.
Irradiation experiments were made in a buffer containing 0.2 M potassium phosphate, pH 7.2, and 0. 1 mM DTE (dithioerythritol). To avoid aggregate formation occuring upon irradiation, it was necessary to use protein concentrations smaller than 5~10~~ protomer.
M
IPTG activity was measured by equilibrium dialysis, according to GILBERT and MULLER-HILL(13). TMS II buffer was used : 0.2 M K Cl, 0. 01 M Mg acetate, 0. 04 M tris/HCl pH 7. 5, 0. 1 mM EDTA and 0. 1 M mercapthoethanol. p4C] IPTG was lo-’ M, giving 500 cpm per 100 ml. Three separated samples were dialyzed for each irradiated solution, the dispersion of the results being less than 5 ‘$. Under these conditions, the measured activity is directly where K is the equilibrium constant of the reaction proportional to Kxn, IPTG and n the determined
OSRAM a band in the a KIPP
number from
t Rep
of IPTG equilibrium
1
Complex
binding sites per dialysis data
Irradiatiomof lac HBO 200 watts mercury between 250 and 400 nm. wavelength range transmitted and ZONEN thermopile.
protomer. K and in the irradiation
n were buffer.
repressor were performed with an lamp. A filter MT0 H 325 a isolated Total incident light energy fluence by the filter was measured with
Fluorescence spectra were recorded with a JOBINYVON spectrofluorimeter modified to correct for fluctuations in lamp intensity(14). Th e a b sorbance of the samples at the exciting wavelength was smaller than 0. 05 to keep the fluorescence intensity proportional to the concentration of fluorescent species.
Vol. 74, No. 2, 1977
Figure upper lower
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
1 : curve curve
A. Relative variation with incident light energy fluence, of -a-*: circular dichroism intensity at 222 nm (A’/&.io) : fluorescence intensity at 340 nm (A,, = 280 nm) binding activity AI/AI, ( -0 -0 - ) for IF/IF ( -D-W) and IPTG irradiated alone. -lac repressor middle curve : fluorescence intensity at 340 nm (hex = 280 nm) IF/IF ( -a - 0 -) and IPTG binding activity AI/AI (-++-) for & repressor irradiated in the presence of o1O-3 M IPTG. I3.
Correlation
IF
curve
- IF
=f(
AI
IF 0 tive fluorescence full circles : lac repressor irradiated
a ROUSSEL-
decrease repressor in
Circular JOUAN
the
and relative irracliated presence
dichroism II apparatus.
;
-
A I)
between
rela-
IO
loss of IPTG binding alone - open circles of 10m3 M IPTG.
spectra
were
recorded
activity. : -lac
at
2°C
with
Tryptophan concentrations were determined according to a procedure derived from SASAKI(l5). The protein was first digested at pH 8 during 5 hours at 3 1 “C, with trypsin and A-chymotrypsin, both at a final concentration of 0. 003 % (w/v). A second proteolytic cleavage with pronase (0.003 % w/v) was performed during 7 hours Guanidinium chloride was used as denatuunder the same conditions. ring agent, at a final concentration of 5 M. After 30 minutes, the fluorescence spectra were recorded. Above 340 nm, where only tryptophan emits, the fluorescence spectrum of the digested protein is identical to that of free tryptophan. Assays on intact lac repressor gave a number of tryptophan residues per protomer equalto 2 f 0. 1, demonstrating that the procedure is quite suitable for this protein.
Results Upon repressor
exhibited
U. V. a rapid
irradiation
(250
decrease
of
692
nm its
< h C 390
fluores.cence
nm), intensity
-lac
:
Vol. 74, No. 2, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
SF CM-‘) IO5
0
’
r
1
0.5
Figure 2 : Scatchard plots for the binding of IPTG to -lac repress0r.r is the fraction of liganded protomers, and Cf is the concentration of free IPTG. The inserted table gives the binding parameters compared with the decrease of fluorescence intensity. A, unirradiated lac repressor, B, irradiated with 2. 1 x lo5 Jmm2 (total incidentlight) C, irradiated with 4 x lo5 Jmm2.
(fig,
1A).
when
excitation
For
The
was
fluorescence was
of photolysis
3~10~
rescence)
of the
wavelength
durations
about
shape
Joules/m’
chosen
longer
blue
when
shift
the
at
than
of incident
a small
observed
spectrum wavelength
and
40
maximum
fluorescence
not
than
295
(corresponding %
of loss
wavelength
was
modified,
longer
10 minutes
light,
of the
was
excited
at
290
to
of of and
nm.
fluo2 and 280
4 nm nm
respectively. Long broad and
and an
band 345
weak
irradiation
band,
spectrum
interfered
(30
with
the
minutes)
a maximum
centered
with
at 345
around
nm.
fluorescence
led
to a new 435-440
Nevertheless,
intensity
nm, this
measurements
at
nm. IPTG
creased
upon
intensity
and
figure of
of
emission
excitation
never
times
8 nm
IB.
binding
irradiation
The
of the
that
of
activity IA).
(fig. the
binding fluorescence
IPTG of
of The
activity
IPTG
to
spectrum
the
decrease are
lac
78)
-lac
linearly
repressor . Similar
repressor of
(Kxn) the
effects
de-
fluorescence
related, leads
also
as to a blue were
shown shift obser-
on
BIOCHEMICAL
Vol. 74, No. 2, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
II ,Fo_ /'
1'0
0.75-
/
T,-T TO
l -•-
I 10
S Incident
light
I 15
energy
flucnce
-3Lx1O5
JxM
f$fg
: Relative variation of the decrease of the fluorescence : fluorescence intensity) and the number of destroyed content) with incident light energy fluence. (T : tryptophan upper part shows the ratio between these two parameters.
phan The
ved
with
irradiated
diation
(and
-lac
therefore
of
In both
IPTG
times
binding
slower
the
same
as
of
bound
the
intensity
No
occurs
change
disappears. markedly
band
indicates
against
photochemical
‘$
the
(fig.
that
the
lA),
two
irra-
IPTG, 5
which
indicates
damages.
parameters
remains
1B).
relative
variation
characteristic of the
of decreased
(fig.
the
of
of
fluorescence conformation
of the the
peptidic
(or
IPTG
of
the
circular bonds.
activity) protein
is
not
affected.
irradiated
straight
75
intensity
repressor
IPTG
of the
Quantitative to
of shows
duration
concentrations
between
1A
until
This
saturating
IPTG
absence
the loss).
fluorescence
correlation
Figure dichroism
of and
of
fluorescence
unliganded
linear in
initial
presence
for
effect the
the
activity
than
a protective However,
the
independent
repressor,
intentrypto-
lac parallel
analysis
repressor lines.
is Binding
of the
shown
on
parameters
694
binding
figure
process 2.
are
of
Scatchard listed
in
IPTG plots
figure
are 2.
BIOCHEMICAL
Vol. 74, No. 2, 1977
The ted
repressor
Figure diated the
protein, protein.
range
determination
was
3 shows
the
relative
and
the
ratio
is
and
the
-lac
at
shorter
the
repressor
310
diation
is
is
nm. due
of the
due
The to
CD
protein
fraction
of
rescence
is
one
the
irra-
two
in in
a large
destruction of
the
of tryptotal
fluo-
the
other
the
modified
tryptophyl of the
photoproduct, close
first
one
a large
the
be is
- always
the
The
of
put
residues
the
upon
absence
change fluorescence.
to
that
irraof varia-
tryptophan
forward
half
weakly
spectrum,
conformational
quenching can
in intensity
3).
of
emit
shoulder
(fig.
does
one
or
by
a local
to
the
indole
residue and
the
tryptophan
is
not
residue
other
tryptophyl
modified,
for
fluorescence
Tyrosines
fluorescence
that
residue
(ii)
each
the
the
explain
of the
that
relative
the fluo-
:
and
group
of
tryptophyl
protein.
- is
fluoresce
at all,
the
in
is
is
same
same
quenched
either
all
by
conformational
modified
in
neither
in
protomers energy
change
all native
and transfer
three
types
tryptophan binding
third
of
protomers
209
modified,
bringing
a quen-
probability
of
ring. (190
second
or one
modified,
for
experiments
hypothesis
209) no
has
longer
reasons
can
should and are
the to
an
equal
emits
fluorescence
identical
to
those
In
this
when given
(ii). The
IPTG
Methods.
of
to
nm,
a small
hypotheses
tryptophyl
fluorescence
produce
responsible
irradiated
in
the
residues.
excludes
in
the
equal
325-380
modification
spectrum
nor
being
irradia-
destroyed
decrease
tryptophyl
tryptophan
modified
protomers
(iii)
to
decrease
decrease
only
ching
the
range
and
Several
the
is that
as
wavelength
wavelengths,
around
(i)
intensity
values
demonstrating slow
and
of tryptophan
these
as
Materials
U. V.
Conclusion In
of the
amount
in
intensity.
Discussion
tion
in
of fluorescence
relative
twice
content
described
loss
time,
residues
rescence
as
between
of irradiation
tophyl
of tryptophan
performed
The
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
be
exist
: intact
protomers
with
in
disagreement
discarded, ones,
protomers
tryptophan with
case,
190 the
with modified.
existence
of
Vol. 74, No. 2, 1977
three
types
the
two
has
been
of
and
15 times linear
plots
presented
equal
to only
for
of
properties,
and
tryptophan
keep
their
binding
that
of
wild
the
of
linear
these
and
are
respect
1.
The
can
in
activity
for
for
A lo0
species
a value
thus
of
n
conclude
irradiated
solutions
to both
fluorescence
and
“dead”
with
to
protomers
should
Scatchard to
We
that residue
type
extrapolate
present
found
two
to
intensity.
protomers with
one
extrapolating
2 are
unaffected
binding
to
fluorescence
types
(10)
A mixture
plots
figure
relative
protomers
one,
A 209.
coworkers
where
equal
Scatchard
two
A 209,
constant
in
the
and
and
a tyrosine
a binding
non
190
by
lower
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
SOMMER
A
replaced
with
that
protomers.
repressors
IPTG
give
BIOCHEMICAL
respect
:
IPTG these
two
properties. It is The
appearance
upon
absorption
band
rescence
quenching
one
intact
between in
being
yet
not
close
available
possible
to
the
the
of SOMMER
taining
only
that of
the the
two wild
tyrosine with
repressors type.
and
not
obvious
of that
transfer of
of
tyrosine modify
the
that
two may the
the
wild
mutants. induce fluorescence
one
type
protein
-lac
to
adequate information
other
mutants
about is
the could
probably
change of
con-
tryptophyl
half
that
replaced
can
replacement
yield
the
observed
a yield
When
would
it
with
built
molecules
the
the
repressor,
authors
conformational quantum
No
a tryptophan
Furthermore, a local
have
with
repressor.
from
The
contradiction
These
these
residue.
assumption.
who
mutants,
from
that
an
fluo-
transfer
requires
of the
apparent (10)
turn
(ii).
that
require
residues.
this
fluorescent
these
in
two
tryptophans.
modified
energy
fluorescence
also
is
photochemically
present
by
it
in
in
coworkers
are
But
is
energy
tryptophan
which
and with
possible
3 would
support
(i) species
it to
structure
or
two
due
figure
of the
(i)
of the
is
to unity
tertiary
and
one
in
eliminate
makes
modified
proximity
Hypothesis results
other
hypotheses fluorescent
nm
protomer
equal
on
between
350
presented is
and
and
each to
probability
orientation
chcrse
of a new
300
results
transfer
to
irradiation
tryptophan
experimental
is
difficult
be
two
by
compared tryptophans
occur, not
a
are
and be
of which
the
the sum
tryptophan might residues.
Vol. 74, No. 2, 1977
BIOCHEMICAL
We vement
of
interact
tryptophan
prevent
does
not
a local
with this
due
destroyed
6, and
repressor
but
that
as
that
tryptophan induces
preventing
IPTG
of binding
another
this
tryptophan,
may
destruction
CD,
of
imply
that
decrease
degradation
rate
its
by the
invol-
modification is
observed
would
CD
been
acti-
amino
last
acid
residue
suggesting
the
is
a very
results
least
indirectly,
of the
strongly the
IPTG,
efficient
least
one
that
the
IPTG
to
tryptophyl of
binding
is
trypto-
of IPTG
of tryptophan.
tryptophan
binding
of
ab-
modification
and
one
differential
binding
modification
that
effector
the
photochemical
photochemical suggest
in
that
of at
the
to bind
(8) ,
fluorescence (17)
that
ability
rate
by
environment
show
the
these
the
results
abolishes
shown
measurements
modifies Our
modifies
photochemical
effector,
direct
process.
sorption(l
phan
the
the
Tryptophan
possibility
excluded
same
It has
residue.
its
not
assumption
the
photosensitized
be
on
process.
Another
photosensitized
an
with
binding and
with
cannot
the
Such
IPTG
change,
it
to
unambiguously
sugar,
directly
Also
residue.
-lac
the
conformational
is
the
interaction.
interact
binding.
conclude
in
directly
may
vity
cannot
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
involved,
All at
process.
Acknowledgements We J.
MILLER
published
for
would
many
like
to
fruitful
thank
Drs.
discussions,
K.
BEYREUTHER
and
communication
and of un-
results.
References (1)
BEYREUTHER,
K.,
ADLER, Proc. Nat. K., ADLER, KLEMM, A. and (1973)
(2)
BEYREUTHER,
(3)
GILBERT,W.
(4) (5)
MCLLER-HILL, MULLER-HILL,
(6) RIGGS, (7)
BARKLEY,
them., 2, and MAXAM, 3, 3581-3584
K., GEISSLER, N. and KLEMM, A. Acad. Sci. USA, 70, 3576-3580 K., FANNING, E., MURRAY, C., GEISSLER, N. (1975) Eur. J. Bio-
491-509 A.
(1973)
Proc.
Nat.
Acad.
Sci.
USA
B. (1975) Prog. Biophys. Mol. Biol. 30, 227-252 B. (1971) Angew. Chem. Int. Ed., lo, 160-171 NEWBY, R. F. and BOURGEOIS, S. (1970) J. Mol.
A. D., 5J, 303-314 M.D., RIGGS, (1975) Biochemistry,
A. D., 14,
697
JOBE, A. 1700-1712
and
BOURGEOIS,
Biol. S.
,
Vol. 74, No. 2, 1977
(8)
LAIKEN,
(9)
SOMMER,
(10)
SOMMER,
(11) (12)
(13) (14) (15) (16)
(17)
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
S. L., GROSS, C. A. and Biol., 66, 143-155 H. , LU, P. and MILLER, lJ, 302a
VON
HIPPEL, J. H.
(1975)
P.
(1972)
Biophys.
J.
Mol.
J.
,
H., LU, P. and MILLER, J. H. (1976) J. Biol. Chem., 251, 3774-3779 SMITH, K. C. and HANAWALT, P. C. (1969) in “Molecular Photobiology”, p. 87, Acad. Press N. Y. MULLER-HILL, B., BEYREUTHER, K. T. and GILBERT, W. (1974) in “Methods in Enzymology”, Vol. XXI, part D, p. 483-487, Acad. Press N. Y. GILBERT, W. and MWLLER-HILL, B. (1966) Proc. Nat. Acad. Sci. USA,%, 1891-1898 TOULME, J. J. and HELENE, C. (1975) Biochemistry, BRUN, F., 2, 558-563 SASAKI, T., ABRAMS, B. and HORECKER, B. L. (1975) Anal. Biochem., 65, 396-404 MATTHEWS, K.S., MATTHEWS, H.R., THIELMANN, H. W. and JARDETZKY, 0. (1973) Biochim. Biophys. Acta. , 285, 159-165 MAURIZOT,J. C. et., to be published.
698
