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PROTEINS: Structure, Function, and Genetics 37:253–263 (1999)
Fluorescence Quenching in the DsbA Protein from
Escherichia coli: Complete Picture of the Excited-State
Energy Pathway and Evidence for the Reshuffling
Dynamics of the Microstates of Tryptophan
Alain Sillen,1 Jens Hennecke,2 Daniela Roethlisberger,2 Rudi Glockshuber,2 and Yves Engelborghs1*
1Laboratory of Biomolecular Dynamics, University of Leuven, Leuven, Belgium
2Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, Zürich, Switzerland
ABSTRACT
The disulfide oxidoreductase DsbA
is a strong oxidant of protein thiols and is required
for efficient disulfide bond formation in the bacterial periplasm. DsbA contains two tryptophans: W76
and W126. The fluorescence of W76 changes upon
reduction of the disulfide bridge, as analyzed previously (Hennecke et al., Biochemistry 1997;36:6391–
6400). The fluorescence of W126 is highly quenched.
The only two potential side chain quenchers are Q74
and N127, and these were replaced by alanine, resulting in a threefold increase in fluorescence intensity.
The fluorescence intensity increase is not due to the
removal of dynamic quenchers but to an increase in
the population with the longest lifetime. In this
report, the possibility of a change in the conformation of W126 is investigated theoretically by using
molecular mechanics and dynamic simulations and
experimentally by using a reaction with N-bromosuccinimide. This reacts preferably with the most exposed microstate of tryptophan, which is responsible for the longest lifetime. The simulations and
the experimental results reveal that the amino acid
replacements allow W126 to increase the population
of its antiperpendicular conformation. The selectivity of the N-bromosuccinimide reaction allows the
visualization of the reshuffling kinetics at exhausting reagent concentration. To the authors’ knowledge, this is the first time that the kinetics of Trp
population reshuffling have been measured. Proteins 1999;37:253–263. r 1999 Wiley-Liss, Inc.
Key words: protein dynamics; molecular mechanics; time-resolved fluorescence; nuclear
magnetic resonance structure; X-ray
structure; N-bromosuccinimide
INTRODUCTION
Internal quenching of tryptophan fluorescence is a phenomenon appreciated among biochemists who are interested in the study of the conformational changes in
proteins.1 The nature of internal quenching of tryptophan
fluorescence in proteins is mainly dynamic. Possible
quenchers in proteins are the side chains of the amino
acids histidine,2,3 cysteine and its disulfide bridge,4 arginine and lysine,5 and the peptide backbone.6 Static quenchr 1999 WILEY-LISS, INC.
ing also is observed in proteins, but it is possible that this
is due to dynamic quenching of very high frequency that
escapes detection by nanosecond (ns) lifetime analysis.7
Some proteins possess tryptophans that are very strongly
quenched, e.g., W126 in DsbA.8 It is of interest to determine the cause of this strong quenching, because the
interaction with the quencher can be used to monitor
conformational changes. Removal of the quencher by sitedirected mutagenesis can reveal the interaction with other
residues and/or substrates and eventually can make the
tryptophan a better reporter group.
DsbA from Escherichia coli is a member of the thioldisulfide oxidoreductase (TDOR) enzyme family and is a
monomeric, periplasmic protein of 21,130 Da (189 amino
acids) required for efficient disulfide bond formation.9,10
TDOR enzymes are involved in numerous processes in
prokaryotic and eukaryotic cells.11–13 All TDOR enzymes
catalyze the formation or reduction of structural, regulatory, or catalytic disulfide bridges in target proteins by
disulfide exchange reactions with their substrates. The
C-X-X-C motif of the active-site is characteristic for all
TDORs. Reduction of the catalytic disulfide bridge in DsbA
has been shown to cause a strong increase in tryptophan
fluorescence.8
The three-dimensional X-ray structure of oxidized DsbA14
has revealed that the enzyme possesses a thioredoxin-like
domain (residues 1–62 and 139–189), a motif that is found
in all known structures of disulfide oxidoreductases.15 The
sequence of the thioredoxin-like domain of DsbA, however,
is only 10% identical with E. coli thioredoxin. DsbA
possesses a second domain (residues 63–138) of unknown
function that is inserted into the thioredoxin motif and
contains the only two tryptophans of DsbA: W76 and
Abbreviations: TDOR, thiol-disulfide oxidoreductase; DTT, dithiothreitol; NBS, N-bromosuccinimide; DTNB, dithionitrobenzoic acid;
IPTG, isopropyl-␤-D-thiogalactoside; DAS, decay associated spectra;
MD, molecular dynamics.
Grant sponsor: Fund for Scientific Research (Belgium/Flanders);
Grant number: G.0242.96; Grant sponsor: ETH Zürich (Switzerland).
Jens Hennecke’s present address is the Structural Molecular Biology Laboratory, Department of Molecular and Cellular Biology, Howard
Hughes Medical Institute, Harvard University, 7 Divinity Avenue,
Cambridge, MA 02138.
*Correspondence to: Yves Engelborghs, Celestijnenlaan 200D, B-3001
Leuven, Belgium. E-mail: yves.engelborghs@fys.kuleuven.ac.be
Received 19 January 1999; Accepted 10 May 1999
254
A. SILLEN ET AL.
Fig. 1. Molscript representation56 of the X-ray
structure of oxidized DsbA from Escherichia coli.
W126. W76 is responsible for the large fluorescence change
upon reduction of the disulfide bridge8 and is located next
to the thioredoxin-like domain of DsbA.14 W126 is located
deeper in the second domain, and its fluorescence is
strongly quenched.
In this study, we investigated the cause of this strong
fluorescence quenching, and we increased the fluorescence
intensity of W126 by site-directed mutagenesis. This made
W126 a good probe with which to monitor possible changes
in the second domain upon reduction of DsbA. A closer look
at the X-ray structure of DsbA revealed that Q76 and N127
are both in close contact with W126 (Fig. 1), within 4.3 Å
and 5.0 Å, respectively. The quenching ability of these
amide groups was interesting to investigate, because some
authors have claimed that amide groups are weak quenchers,4 others have claimed that amide groups are not
quenchers at all,16 and others have claimed that amide
groups can quench in special circumstances with the aid of
other amide groups or with the aid of the peptide backbone.6
MATERIALS AND METHODS
Materials
Dithiothreitol (DTT), N-acetyltryptophanamide, and
tryptophan were obtained from Sigma Chemical Co. (Buchs,
Switzerland) The buffer solution used was a 0.05 M
KH2PO4 buffer adjusted with NaOH to pH 7.0. The ionic
strength of the solutions was always 0.1 M adjusted with
KCl. All buffer reagents were of analytical grade and were
obtained from either Sigma, Janssen Chimica (Geel, Belgium), or Fluka (Buchs, Switzerland). Ampicillin, dithionitrobenzoic acid (DTNB), and polymyxin B sulfate were
purchased from Sigma. Isopropyl-␤-D-thio galactoside
(IPTG) was from AGS GmbH (Heidelberg, Germany),
DE52- and CM52-cellulose were purchased from Whatman (Maidstone, United Kingdom), and Phenyl-Superose
HR 10/10 column was obtained from Pharmacia (Uppsala,
Sweden). All other chemicals were from Merck (Darmstadt, Germany) or Fluka and were of the highest purity
available. Solutions were filtered (0.22 µm), and assayed
for spectral purity.
Mutagenesis
For construction and expression of the DsbA variants
Q74A and N127A, the phasmid pDsbA2 was used, harboring the dsbA gene under control of the trc-promotor.17
Site-directed mutagenesis was performed according to
Kunkel et al.18,19 by using the helper phage M13KO720
and the kit supplied by Bio-Rad (Hercules, CA). The
following oligonucleotides were used: Q74A: 58-GCC ATC
GCC ACT GCC CAT GCC GCT GTC AGG TCT TTG CCC
AGG TC-38 N127A: 58-GAG ATT TCA CCA CAA AGC TAG
CCC ACG CTG CAT CAT ACT CTT CAC CTT TA-38.
The DsbA variants Q74A/W76F, W76F/N127A, and
Q74A/W76F/N127A were constructed by using polymerase
chain reaction (PCR). By using the plasmid pDSBA2 as a
template, the 58-oligonucleotide (Q74A/W76F) 58-CGC ATG
GAA TTC AGA TCT GAC TGC GGC ATT TGC TGT GGC
255
FLUORESCENCE QUENCHING IN DSBA
GAT GGC G-38 (the BglII restriction site is indicated in
italics) and the 38-oligonucleotide 58-CTG CGC ACC GGT
ACC GCC ACC TTT TTT CTC GGA CAG ATA TTT C-38
parts of the dsbA gene were amplified by PCR, and a 316
base pair (bp) fragment was cloned through the BglII and
PstI restriction sites into the plasmid pDsbA2, generating
pDSBA2-Q74A/W76F. The plasmid pDSBA2 Q74A/W76F/
N127A was constructed essentially in the same manner,
but pDSBA2-N127A was used as the template for PCR. For
construction of pDSBA2-W76F/N127A, the plasmid
pDSBA2-N127A was used as the template, and the 58oligonucleotide (W76F) 58-CGC ATG GAA TTC AGA TCT
GAC TCA GGC ATT TGC TGT GGC GAT GGC G-38 (the
BglII restriction site is indicated in italics) and the same
38-oligonucleotide described above were used for the PCR.
Again, the 316-bp fragment was cloned through the BglII
and PstI restriction sites. Mutants were identified by
restriction analysis and verified by sequencing of the whole
mutated genes using the T7 sequencing kit (Pharmacia).
intermediate frequency of 455 kHz by external crosscorrelation and is then filtered and amplified further. The
phase shift is measured in the low-frequency domain (700
Hz) by using a second cross-correlation step. In this way,
fluorescence lifetime measurements are performed by measuring the phase shift of the modulated emission at 50
frequencies ranging from 1.6 MHz to ⬇1 GHz. Either
N-acetyltryptophanamide (in water filtered by using the
MilliQ system; Millipore Corp., Bedford, MA) with a
fluorescence lifetime of 3.059 ns or p-terphenyl in cyclohexane
with a lifetime of 1.04 ns26 was used as a reference fluorophore.
The measured phase shifts (␾) at a modulation frequency (␻)
of the exciting light are related to the fluorescence decay in the
time domain, as described previously.27 Data analysis was
performed as described by Vos et al.25
Quantum yields were determined relative to tryptophan
in water according to the method of Parker and Rees.28
Qprot ⫽
Expression and Purification of DsbA Variants
For expression of DsbA variants in the bacterial
periplasm, the E. coli strain THZ221 was used. The
methods used have been described previously.8,17 All DsbA
variants were obtained in the oxidized form after purification, as shown by the lack of free thiol groups,22 and their
correct molecular mass was confirmed by MALDI mass
spectrometry.
Ultraviolet Absorption Measurements
Ultraviolet absorption was measured on a Kontron 940
spectrophotometer (Uvikon). The molar absorption coefficients at 295 nm are calculated by taking the ratio of the
absorbance at 295 nm and at 280 nm multiplied by the
molar absorption coefficients at 280 nm obtained from
Hennecke et al.8
Steady-State Fluorescence Measurements
Steady-state fluorescence was measured with an SPEX
spectrofluorometer (Fluorolog 1691), as described by Sillen
et al.,23 with excitation and emission slits providing band
passes of 7.2 nm and 3.6 nm, respectively. The cuvette
holder was thermostated at 22°C. The excitation wavelength was 295 nm to ensure that the measured fluorescence was due only to tryptophan fluorescence.
Fluorescence Lifetime Data Measurements
Fluorescence lifetime data were determined by using an
automated multifrequency phase fluorometer. The instrument is similar to the one described by Lakowicz et al.,24
except for the use of a high-gain photomultiplier (Hamamatsu H5023) instead of a microchannel plate. The detection part was described previously by Vos et al.25 The
excitation source consists of a mode-locked, titaniumdoped sapphire laser (Tsunami; Spectra Physics) pumped
by a Beamlok 2080 Ar⫹-ion laser (2080; Spectra Physics).
After frequency tripling (GWU; Spectra Physics), the
excitation wavelength is 295 nm. A single harmonic component of the exciting light pulse train is converted first to an
兰I
兰I
Prot ATrp
QTrp,
(1)
Trp AProt
where 兰I is the integrated intensity over the wavelength
region 300–450 nm, A is the absorbance at 295 nm, and the
quantum yield (QTrp) for tryptophan in water is 0.14.29
Decay-Associated Spectra
Decay-associated spectra were constructed by multiplying the intensity fraction with the intensity of the emission
spectra at the respective wavelengths.30 A log-normal
function31 is fitted to the associated intensities to obtain
the decay-associated spectra:
3
I(␭) ⫽ Im exp ⫺
ln 2
ln ␳
2
ln2
1
(a ⫺ 1/␭)
24
(a ⫺ 1/␭m)
.
(2)
Here, Im ⫽ I(␭m) is the maximal fluorescence intensity; ␭m
is the wavelength of the band maximum; ␳ ⫽ (1/␭m ⫺
1/␭⫺)/(1/␭⫹ ⫺ 1/␭m) is the band asymmetry parameter; a ⫽
1/␭m ⫹ (1/␭⫹ ⫺ 1/␭⫺) ␳/(␳2 ⫺ 1) is the function-limiting
point; and ␭⫹ ⫽ 107/(0.830 · 107/␭m ⫹ 7,071) and ␭⫺ ⫽
107/(1.1768 · 107/␭m ⫺ 7,681) are the wavelength positions
of half-maximal amplitudes.
The average radiative rate constant is calculated by
dividing the quantum yield by a wavelength-independent
amplitude average lifetime:32
7kr8 ⫽
Q
⌺␶i␣i
.
(3)
With ⌺␶i␣i the average lifetime, ␣i is a wavelengthindependent amplitude fraction and is defined as32,33
␣i ⫽
兰I
0i(␭)
d␭
⫽
l⫽n
兺兰I
l⫽1
0l(␭)
d␭
兰 I (␭) d␭
.
I
(␭)
d␭
兰
0i
0
(4)
256
A. SILLEN ET AL.
The fluorescence intensity at time zero (I0) of the corresponding lifetime is integrated over all wavelengths and is
then normalized.
Calculation of the Change in Fluorescence
Intensity Due to Static and Dynamic Quenching
and by a Change in the Population of
Microconformations
We suggest splitting the ratio of the quantum yield of
different variants relative to a reference protein [e.g., wild
type (WT)] into a factor (fkr) representing the change in kr,
or homogenous static quenching, a factor (fPR) reflecting
heterogenous static quenching or population reshuffling,
and a factor (fDQ) representing pure dynamic quenching:32
Q
Q0
⫽
7kr8 ⌺␣i␶i
7kr08⌺␣0i␶0i
⫽
7kr8 ⌺␣i␶0i ⌺␣i␶i
7kr08 ⌺␣0i␶0i ⌺␣i␶0i
⫽ fkr ⫻ fPR ⫻ fDQ.
(5)
The factor fPR is affected by static quenching only if the
static quenching is heterogenous. If there is static quenching and an increase in the fluorescence due to population
reshuffling, then fPR is the minimum increasing factor of
fluorescence intensity due to a change in microconformations.
Energy Map
To characterize the possible conformations of tryptophan, a minimum perturbation map was calculated.34
Minimum perturbation mapping of W126 was performed
by using the CHARMM22 package.35 Map calculation was
done by using the all-hydrogen CHARMM22 parameter
and topology file. The system contained no water. Minimum perturbation maps36 are calculated by fixing the Trp
side chain at a particular ␹1, ␹2 point and allowing the
residues nearby Trp to conformationally relax to achieve
an energy minimization in which all of the other residues
are fixed to their crystallographic or nuclear magnetic
resonance (NMR) coordinates. The nearby residues included K70, D71, L72, T73, Q74, A75, W76, A77, V78, A79,
A81, L82, A105, R109, F112, I117, E120, E121, Y122,
D123, A124, A125, N127, S128, F129, V130, K131, K132,
S133, L134, V135, and A136. The minimization procedure,
used at each grid point on the ␹1, ␹2 map, consists of 100
steps by the steepest descent method and 1,000 steps by
the Powell method with a tolerance of 0.1. This is repeated
in 10° steps over the whole angular space of ␹1 and ␹2.
Throughout this article, ␹1 is defined as the torsion angle
around C␣-C␤ by the bond connectivity N-C␣-C␤-C␥, and ␹2
is defined as the torsion angle around C␤-C␥ by the bond
connectivity C␣-C␤-C␥-C␦1. These maps display areas where
Trp is in an energetically favored position.
Molecular Dynamics
The three-dimensional X-ray structure of oxidized DsbA14
was obtained from the Brookhaven Protein Data Bank.37
The GROMOS 8738 package was obtained from Biostructure S.A. (France). The input files for the GROMOS 87
package were generated by using the program WHATIF,39
which also was used to make the mutations in the threedimensional NMR structure. The structures were placed
in a truncated octahedral box of SPC water40 with the
counter ions (Cl⫺ and Na⫹), where a minimum distance of
7 Å was kept between the protein and the border of the box.
The energies of the protein and the water were then
minimized for 500 steps by using a steepest-descent algorithm.41 The velocities of the atoms were assigned following a Maxwellian velocity distribution at 100 K. The
system was warmed up to 300 K in two consecutive steps of
1.3 ps in total, and a free molecular dynamics (MD)
simulation was performed for 200 ps using a constant
pressure of 1 MPa and a constant temperature of 300 K.
The temperature of the protein and the solvent were
coupled separately to a water bath42 using a coupling
constant of 0.1 ps. The pressure was kept constant by
coupling to an external pressure bath42 with a coupling
constant of 0.5 ps. The conditions of the MD simulation
were the following: the time step employed was 2 fs, the
integration of the equations of motion and energy were
done by using a leap-frog algorithm included in the
GROMOS package, the bond lengths were constrained by
using the SHAKE routine,43 and cut-offs of 8 Å for nonbonded interactions (van der Waals interactions) and 11 Å
for the electrostatic interactions were used. For analysis,
the coordinates were saved every ps. The calculations were
performed by using an Indigo 2 workstation (Silicon
Graphics) equipped with an MIPS R10000 processor. The
data were analyzed by using the programs WHATIF42 and
SIMLYS.44
Reaction With N-Bromosuccinimide
The accessibility of the tryptophan conformation was
probed with N-bromosuccinimide (NBS) according to the
method of Spande and Witkop,45 as described by Sillen et
al.23 Measurements were done at pH 7.0 (0.1 M sodium
phosphate buffer). Titration of DsbA W76F/N127A/Q74A
with increasing amounts of NBS was performed by adding
aliquots of a 4.9-mM stock solution of NBS to the cuvette
containing the protein (95 µM), after which the changes in
the fluorescence lifetimes and corresponding amplitude
fractions were monitored. In a second experiment, the
steady-state fluorescence intensity was monitored after
the addition of a tenfold excess of NBS over DsbA W76F/
N127A/Q74A mutant concentration. A fluorescence decrease followed by a fluorescence increase was observed.
The curve was fitted with the following equation:
Fss(t) ⫽ A(1 ⫺ exp (⫺krect)) ⫹ B exp (⫺kNBSt),
(6)
where A is the amplitude of fluorescence recovery, krec is
the rate constant of recovery that is attributed to population reshuffling, kNBS is a rate constant of reaction with
NBS, and B is the amplitude of the fluorescence decrease,
omitting the first 4 seconds to avoid mixing artifacts.
FLUORESCENCE QUENCHING IN DSBA
257
Fig. 2. Decay-associated spectra of DsbA variants in the oxidized
state (ox) at 22°C and pH 7.0 (excitation, 295 nm). The deviation at low
wavelength between the calculated and the measured spectra is due to
the contribution of scattering. Circles, short lifetime ␶1; squares, middle
lifetime ␶2; triangles, long lifetime ␶3; solid lines, measured emission
spectra; dotted lines, sum of the decay-associated spectra.
RESULTS
Fluorescence Spectra and Quantum Yield of the
DsbA Variants
suggesting the absence of conformational changes in the
second domain upon reduction of the disulfide bond.
Analysis of the fluorescence spectra (Fig. 2) and the
quantum yields (Table I) of the DsbA variants W76F,
W76F/N127A, W76F/Q74A, and W76F/N127A/Q74A in
both oxidized and reduced states reveals that the variant
W76F has a strongly quenched tryptophan fluorescence
and that removing N127 or Q74 leads to a fluorescence
increase in the oxidized state by 15% and 123%, respectively. This suggests that the amide groups in Q74 and
N127 indeed are potential quenchers of tryptophan fluorescence.46 Reducing the disulfide bridge, on the other hand,
does not significantly change the fluorescence properties of
the DsbA variants in which W74 is removed, strongly
Time-Resolved Fluorescence Parameters
The fluorescence decay of W126 in the variants W76F,
W76F/N127A, W76F/Q74A, and W76F/N127A/Q74A was
measured at emission wavelengths ranging from 330 nm
to 380 nm in 10-nm intervals. A single or double exponential fit of the data yielded unacceptably high values of ␹R2
and a significant nonrandomness in the autocorrelation
function of the weighted residuals as a function of the
frequency. The best fit with lowest ␹R2 and no systematic
deviations in the autocorrelation function of the weighted
residuals was obtained with a triple exponential fit. Different starting values for the fitting procedure resulted in the
258
A. SILLEN ET AL.
TABLE I. Molar Absorption Coefficients, Quantum Yields,
Average Lifetimes, and Radiative Rate Constants of the
DsbA Variants†
⑀295 (M⫺1 cm⫺1)
Variant
W76F ox
W76F/N127A ox
W76F/Q74A ox
W76F/N127A/
Q74A ox
N127A ox
Q74A ox
W76F red
W76F/N127A red
W76F/Q74A red
W76F/N127A/
Q74A red
Q
7␶8␣
7kr8
(ns) (ns⫺1)
2,656 ⫾ 496
2,572 ⫾ 247
2,442 ⫾ 166
0.0132 ⫾ 0.003 0.26 0.051
0.0152 ⫾ 0.002 0.29 0.052
0.0295 ⫾ 0.002 0.64 0.046
2,877 ⫾ 143
6,172 ⫾ 320
6,057 ⫾ 30
2,999 ⫾ 420
2,734 ⫾ 141
2,627 ⫾ 540
0.036 ⫾ 0.005
0.036 ⫾ 0.002
0.046 ⫾ 0.008
0.0118 ⫾ 0.003
0.0147 ⫾ 0.001
0.0291 ⫾ 0.004
2,718 ⫾ 295
0.0409 ⫾ 0.012 0.89 0.046
0.86
0.77
0.89
0.27
0.28
0.61
0.042
0.047
0.047
0.044
0.053
0.048
molar absorption coefficient; Q, quantum yield; 7␶8␣
(ns), average lifetime in nanoseconds; 7kr 8 (ns⫺1 ), average radiative
rate constant.
†⑀
295
(M⫺1
cm⫺1 ),
same end values. Single decay analysis did not show a
systematic increase in the value of the lifetimes at higher
wavelengths, indicating that relaxation processes of the
environment influencing the lifetimes are unlikely. To
improve the recovery of the decay parameters, a global
analysis of all of the phase measurements at the different
wavelengths was performed. The result of this global fit is
summarized in Table II. All of the DsbA variants have
approximately the same fluorescence lifetimes: Only the
amplitude fractions change in the different variants.
Energy Mapping
Minimum perturbation map calculations of W126 were
performed on the energy minimized average NMR structure (PDB ID:1A23).47 The energy map of W126 in WT
(Fig. 3) shows only two stable conformations for the
tryptophan side chain, i.e., ␹1 ⫽ 169° and ␹2 ⫽ 37° (the
perpendicular conformation) is the minimum energy conformation that corresponds exactly to the minimized average NMR conformation, and the second minimum is at
␹1 ⫽ 129° and ␹2 ⫽ ⫺123° (the antiperpendicular conformation) and has an energy that is 60.3 Kcal/mol higher; this
second minimum is relatively close to the X-ray conformation48 (PDB ID: 1A2J), with ␹1 ⫽ 178.3° and ␹2 ⫽ ⫺99.6°.
There are 17 out of the 20 NMR structures that position
W126 in the perpendicular conformation and three structures that position W126 in the antiperpendicular conformation. Apparently, the two conformers are possible in the
solution; however, one minimum is preferred, and the
other is preferred in the crystal form (these experimental
facts prove that the absolute value of the energy difference
of 60 Kcal/mol is largely overestimated, because calculations are done at 0°K for a single molecule; see Discussion).
The energy map of the W76F variant has the same two
regions, with the minimum at ␹1 ⫽ 150° and ␹2 ⫽ 60° and
the second minimum at ␹1 ⫽ 130° and ␹2 ⫽ ⫺110°, with an
energy of 70.4 kcal/mol higher. The energy map of the
W76F/Q74A variant also has two stable conformations;
however, here, the perpendicular conformation (␹1 ⫽ 169°
and ␹2 ⫽ 77°) is only 1.5 kcal/mol higher in energy than the
other minimum (␹1 ⫽ 139° and ␹2 ⫽ ⫺103°).
Molecular Dynamics Simulations
MD simulations in water were performed on the W76F/
Q74A variants of DsbA: one with the starting structure of
W126 in the perpendicular conformation (␹1 ⫽ 169° and
␹2 ⫽ 77°) and the other in the antiperpendicular position
(␹1 ⫽ 139° and ␹2 ⫽ ⫺103°). The ␹1 and ␹2 angles were
monitored during a 200-ps simulation. The only intention
of such a short simulation is to relax the immediate
environment of the tryptophan residues. In both simulations, the ␹ angles remained practically in their starting
position. The distance between the CE3 atom and the C
atom (PDB atom notation) of W126 is monitored during
the MD simulation and averaged. Charge transfer from
the excited indole moiety to the carbonyl group of the
peptide backbone is probably the main nonradiative pathway in proteins.6 In the perpendicular position, the CE3
atom of W126 has a high frequency of approaching (3.9 Å)
the carbonyl group of the backbone, whereas, in the
antiperpendicular position, the distance is greater (4.3 Å),
and the frequency is lower.
Selective Quenching of Fluorescence by Reaction
With NBS
To analyze the orientation of W126, its reaction with
NBS was studied. NBS reacts irreversibly with tryptophan, generating a totally nonfluorescent oxindole product.49 For NBS to react with Trp, it is essential that the CG
and CD1 atoms are solvent-accessible.50 This is the case in
the antiperpendicular conformation (Table III). In the
perpendicular conformations, these atoms are shielded far
more from the solvent. Therefore, the reaction with NBS
should be able to report on the conformation of W126. The
result of the reaction with low NBS concentration is shown
in Figure 4. The amplitude fraction of the largest lifetime
decreases strongly, whereas the amplitude fraction of the
middle lifetime increases. The amplitude fraction of the
smallest lifetime decreases to only a limited extent. Thus,
the conformation responsible for the largest lifetime is the
most reactive toward NBS, whereas the conformation of
the middle lifetime is not reactive or reacts very little to
NBS. The smallest lifetime shows only a limited reactivity.
Also, the steady-state fluorescence decreases strongly upon
reaction with low NBS concentration (Fig. 5); however,
upon exhaustion of NBS, the fluorescence intensity increases again, indicating the reappearance of a long lifetime with a rate constant of 0.0142 ⫾ 0.0002 s⫺1 (Fig. 5,
inset b). This process of fluorescence recovery is attributed
to the reshuffling of the microstates. The calculated rate
constant is only an estimate of the recovery rate, because
the mechanism of fluorescence recovery potentially is more
complicated due to the different accessibilities and reshuffling rates of the three microstates of tryptophan.
259
FLUORESCENCE QUENCHING IN DSBA
TABLE II. Lifetimes, Wavelength Independent Amplitude Fractions, and ␹R2 as Obtained by Global Analysis and DAS of
the Fluorescence Decay of DsbA and its Variants at pH 7.0 in the Oxidized and Reduced States†
␣1
␶1 (ns)
␣2
␶2 (ns)
␣3
␶3 (ns)
␹R2 a
0.94 ⫾ 0.04
0.92 ⫾ 0.02
0.77 ⫾ 0.02
0.75 ⫾ 0.02
0.46 ⫾ 0.02
0.41 ⫾ 0.02
0.93 ⫾ 0.02
0.93 ⫾ 0.03
0.80 ⫾ 0.01
0.75 ⫾ 0.02
0.14 ⫾ 0.01
0.12 ⫾ 0.01
0.14 ⫾ 0.01
0.13 ⫾ 0.01
0.17 ⫾ 0.02
0.18 ⫾ 0.02
0.14 ⫾ 0.03
0.12 ⫾ 0.01
0.14 ⫾ 0.01
0.15 ⫾ 0.01
0.050 ⫾ 0.001
0.037 ⫾ 0.005
0.07 ⫾ 0.008
0.05 ⫾ 0.004
0.48 ⫾ 0.02
0.49 ⫾ 0.02
0.06 ⫾ 0.01
0.03 ⫾ 0.008
0.06 ⫾ 0.01
0.04 ⫾ 0.02
1.81 ⫾ 0.03
1.33 ⫾ 0.2
0.83 ⫾ 0.1
1.03 ⫾ 0.1
1.03 ⫾ 0.03
1.00 ⫾ 0.03
1.73 ⫾ 0.1
1.49 ⫾ 0.2
0.78 ⫾ 0.1
0.77 ⫾ 0.3
0.01 ⫾ 0.04
0.04 ⫾ 0.02
0.15 ⫾ 0.02
0.20 ⫾ 0.02
0.06 ⫾ 0.01
0.10 ⫾ 0.005
0.01 ⫾ 0.02
0.04 ⫾ 0.03
0.15 ⫾ 0.01
0.21 ⫾ 0.002
3.94 ⫾ 0.01
3.16 ⫾ 0.1
3.07 ⫾ 0.06
3.57 ⫾ 0.06
3.21 ⫾ 0.1
3.28 ⫾ 0.1
3.96 ⫾ 0.1
3.10 ⫾ 0.1
3.09 ⫾ 0.1
3.53 ⫾ 0.4
3.9
3.8
3.1
2.4
2.3
3.6
2.8
3.1
3.5
2.6
Variant
W76F ox
W76F/N127A ox
W76F/Q74A ox
W76F/N127A/Q74A ox
N127A ox
Q74A ox
W76F red
W76F/N127A red
W76F/Q74A red
W76F/N127A/Q74A red
†␣,
wavelength independent amplitude fraction; ␶, lifetime; ox, oxidized; red, reduced.
high ␹R2 is due to the very low intensity. (See the quantum yields.)
aThe
Fig. 3. Calculated energy map of W126 (␹1 and ␹2) in DsbA wild type (WT) and W76F/Q74A
variant. Energy is expressed in kcal/mol and is relative to the lowest energy.
TABLE III. Accessible Surface Å2
Calculated on the Basis of the Nuclear
Magnetic Resonance Structure of Reduced
DsbA of Reactive Atoms of W126 in DSBA
W76F/Q74A With N-Bromosuccinimide
Atom
Perpendicular
Antiperpendicular
CG1
CD1
Total
2.49
3.02
5.51
1.32
8.63
9.95
DISCUSSION
Quenching Analysis
Compared with Trp in solution, which has a quantum
yield of 0.14, the fluorescence of W126 is highly quenched
in both the oxidized state (Q ⫽ 0.013) and the reduced
state (Q ⫽ 0.012) of DsbA. This seems to be due largely to
an increase of dynamic quenching, because the apparent
radiative rate constant is the same as the radiative rate
constant of tryptophan, 0.053 ns⫺1,51 whereas the nonradiative rate constant is 3.8 ns⫺1 compared with 0.33 ns⫺1 for
Trp in solution. We therefore looked for dynamic quenchers in the neighborhood of W126. The only two candidates
within collisional distances were amide groups of Q74 and
N127. The quenching constants related to Q74 and N127
are calculated in Table IV. Replacing Q74 and N127 by
alanine indeed reduced the nonradiative rate constant to
lower values, and the quenching constants of Q74 and
N127 clearly are additive in the variant W76F, indicating
parallel pathways of decay. The remaining questions are 1)
how does N127 and Q74 quench the fluorescence of W126,
and 2) why is the remaining nonradiative rate constant
still quite high (1.12 ns⫺1)? Inspection of the lifetime data
(Table II) and detailed quenching analyses (Table V) reveal
that the lifetimes themselves hardly change upon replacement of Q74 or N127. Upon removal of the amide of Q74,
there is a decrease in intensity due to static quenching of
14% (fkr), and replacement of N127 increases the intensity
by only 2%. Replacement of Q74 or N127 has approximately the same effect on dynamic quenching of the
fluorescence of W126; in both cases, the intensity decreases by ⫾20%. This indicates that amide groups are not
260
A. SILLEN ET AL.
TABLE IV. Rate Constant of Fluorescence Decay, Intrinsic
Fluorescence Decay Rate, and Apparent Dynamic
Quenching Caused by N127 and Q74
Variant
7␶8␣
(ns)
W76F/N127A/
Q74A
W76F/Q74A
W76F/N127A
W76F
0.86
1.16
0.64
1.56
0.29
3.44
0.26 3.84a and 3.84b
k
(ns⫺1)
kint kq(N127) kq(Q74)
(ns⫺1) (ns⫺1)
(ns⫺1)
1.16
1.16
1.16
1.16
—
0.4
—
0.4
—
—
2.28
2.28
k, fluorescence decay rate constant; kint, intrinsic fluorescence decay
rate constant; kq, apparent dynamic quenching constant.
aexperimentally observed.
bcalculated on the basis of the single variants.
TABLE V. Relative Quenching Analysis: Static
Quenching, Dynamic Quenching, and Decrease in the
Fluorescence Intensity of W126 by the Change of
Microconformations With W76F as Reference State†
Fig. 4. N-bromosuccinimide (NBS) titration of DsbA at pH 7.0 and
22°C W76F/Q74A/N127A ox at a protein concentration of 95 µM. The
change in the amplitude fraction (␣i) at 340 nm is given as a function of
initial NBS concentration. Circles, short lifetime ␶1; squares, middle
lifetime ␶2; triangles, long lifetime ␶3.
Variant
Q/Q0
fkr
fPR
fDQ
W76F/N127A
W76F/Q74A
W76F/N127A/Q74A
1.15
2.23
2.73
1.05
0.93
0.82
1.35
3.16
3.76
0.81
0.76
0.88
†f
kr , static quenching; fPR , change of microconformations; fDQ ,
dynamic quenching.
lated. The increase in fluorescence intensity due to a
change in microconformations can be calculated by fPR. A
similar phenomenon in which a thermally induced increase in fluorescence intensity of Trp-X peptides is due to
the higher population of microconformation with the longer
lifetime has been reported previously.52 Another (and
apparently the most important) dynamic quencher in the
vicinity of tryptophan is the carbonyl group of the peptide
bond,6 which is very close (3.9 Å) to the indole group in the
perpendicular conformation and, thus, is responsible for
the short lifetime.
Molecular Mechanics and Dynamics
Fig. 5. The change in the steady-state fluorescence intensity on
reaction of NBS with the variant W76F/Q74A/N127A ox as function of
time. Inset b: Fitting of Equation 11 to the fluorescence intensity (see
text).
direct quenchers of tryptophan fluorescence. The cause of
the strong quenching of W126 is due to the high population
of the microconformation with the shortest lifetime. Replacement of Q74 or N127 makes it possible for microconformations with higher lifetimes to become more popu-
The microconformation with the shortest lifetime is 95%
populated. Replacement of N127 increases the population
of the longest lifetime with 4%, replacement of Q74
increases the population of the longest lifetime with 16%,
and replacements of both increase with the sum (20%). To
determine whether it is possible for Trp to change its
conformation, an energy map was calculated. The energy
map calculations reveal that there are two energy minima
of W126 in DsbA in both the WT and the W76F/Q74A
variant. The energy difference between the two minima is
very large in the WT and W76F variant, but the energy
fluctuations of the MD simulations of both conformations
are even larger than the energy difference of the two
minima. This indicates that energy maps provide only a
qualitative idea of the possible energy minima. They also
are made at 0°K rather than at room temperature, and the
backbone of the protein is fixed in these energy maps. All of
this also could explain why there are only two energy
minima on the map although three lifetimes have been
261
FLUORESCENCE QUENCHING IN DSBA
Fig. 6. Scheme 1: The scheme of the total excited-state energy pathway in DsbA. k, Rate
constant of fluorescence decay; kint, intrinsic fluorescence decay rate; kQ, apparent dynamic
quenching; Q74 and N127, side-chain quenchers; W126 and W76, tryptophans.
measured. The middle lifetime must result from another
conformation that was not highly populated in the experimentally determined structures or in the calculations
(compare with ⫾5% in the fluorescence measurements).
However, it is reassuring to note that, in the X-ray
structure, one energy minimum is populated (antiperpendicular) and the other is populated in the NMR structure
(perpendicular).
To investigate the possibility of fluorescence quenching
by the carbonyl group of the peptide bond in both microconformations, an MD simulation was performed, and the
distance between the CE3 of the indole group and the
carbonyl carbon of the peptide bond was monitored. In the
MD simulation, W126 remains in the original conformations. In the perpendicular conformation, the indole group
is closer to the carbonyl of the peptide group compared
with the antiperpendicular conformation. Thus, the perpendicular conformation is quenched more than the antiperpendicular and, thus, is associated with the lowest lifetime.
Linking of the Conformations With the Lifetimes
NBS reacts preferably with the tryptophan that has the
most exposed pyrrole ring. Analysis of the NMR structure
in the reduced DsbA reveals that, in the antiperpendicular
conformation, the pyrrole ring of W126 is the most exposed. Lifetime determinations of DsbA W76F/N127A/
Q74A reacting with increasing amounts of NBS reveal that
the amplitude fraction of the longest lifetime decreases the
greatest. Thus, reaction with NBS identifies the longest
lifetime with the antiperpendicular conformation (␹1 ⫽
139° and ␹2 ⫽ ⫺103°). Also, in the steady-state experiment, at exhausting concentration of NBS (when the
modified Trp redistributes over the different microconformations), a fraction of the long lifetime reappears and
explains the recovery of the fluorescence intensity (Fig. 5).
After some time, the limited excess of NBS has disappeared due to reaction and to hydrolyses of NBS.
The MD simulation of the variant W76F/Q74A reveals
that the carbonyl carbon of the backbone of W126 is closer
in the perpendicular conformation (␹1 ⫽ 169° and ␹2 ⫽ 77°)
Consequently, the lifetime of this conformation may be
lower. Thus, this conformation is linked to the smallest
lifetime and/or the middle lifetime.
Influence of Reduction on the Second Domain
in DsbA
Upon reduction of DsbA, there are no changes in the
fluorescence properties of W126. Changing W126 or the
environment of W126 has an influence on the fluorescence
of W76 that is different in the oxidized state and the
reduced state. Upon oxidation, a static quenching is removed for W76 if N127 or Q74 are changed; whereas, if
W126 is replaced by a Phe, then the static quenching is
removed upon reduction. This is an additional influence of
the oxidation state of the first domain on the W76 fluorescence that depends on the mobility of the W76 in the
second domain.
Overall Scheme of the Quenching in DsbA
The overall scheme of rate constants (Fig. 6) provides a
picture of the fluorescence decay pathway of the two
tryptophans in DsbA. The scheme of the rate constants
that effect the fluorescence of W76 is taken from Hennecke
et al.8 The other rate constants are taken from Table IV.
CONCLUSIONS
The increase in fluorescence intensity of W126 upon
removal of the amides is not due to the removal of
collisional quenchers but to a reshuffling of microstates
toward a population that is less quenched. The high knr in
the triple mutant is due to the fact that the lowest lifetime
is still highly populated (75%). The origin of this lowest
lifetime is that the conformation associated with this
lifetime is very close to the carbonyl group of the peptide
bond. The amide groups do not quench tryptophan fluorescence directly.6,16
Reduction of the disulfide bridge does not seem to lead to
important conformational changes in the second domain of
DsbA, because neither W76F nor the triple mutant showed
any fluorescence change upon reduction. Our results indicate that the multiple exponential fluorescence decay is
262
A. SILLEN ET AL.
caused by multiple microconformations of tryptophan in
the protein matrix53 that slowly interchange from one
conformation to the other, and not by the existence of
multiple acceptors of fluorescence emission.54 Energy transfer to different acceptors is a decay process with parallel
paths from the same source; therefore, the decay rates are
additives that give rise to only one exponential decay.
Other possible explanations for the nonexponential decay of tryptophan fluorescence in proteins are not applicable to the fluorescence of W126. If the nonexponential
decay originates from, e.g., solvent relaxation,55 then
reaction with NBS would not affect one lifetime more than
the others, and, as a result, the steady-state fluorescence
would not increase after the time when the NBS is
exhausted. The same reasoning is applicable to any explanation for the nonexponential decay of tryptophan fluorescence that does not take into account the conformational
heterogeneity of the tryptophan.
Our NBS experiments provide an idea of the time scale
during which large amino acids like tryptophan that are
buried partially in the protein matrix change their conformation. In this protein, the time scale of the process is in
the seconds range. To our knowledge, this is the first report
that has measured the kinetics of Trp population reshuffling.
ACKNOWLEDGMENTS
The authors thank Dr. Dı́az (Leuven) for his help with
the programs SIMLYS and WHATIF/GROMOS and P.
James (Zürich) for performing mass spectrometry.
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