close

Вход

Забыли?

вход по аккаунту

?

226

код для вставкиСкачать
PROTEINS: Structure, Function, and Genetics 40:112–125 (2000)
Aromatic Interactions in Homeodomains Contribute to the
Low Quantum Yield of a Conserved, Buried Tryptophan
Vikas Nanda* and Ludwig Brand
Department of Biology, Johns Hopkins University, Baltimore, Maryland
ABSTRACT
Trp 48, a conserved, buried residue
commonly found in the hydrophobic core of homeodomains, has an unusually low fluorescence quantum
yield. Chemical denaturation of Drosophila homeodomains Engrailed and Antennapedia(C39S) result in a
four-fold increase in quantum yield, while unfolding
of Ultrabithorax causes a twenty-fold enhancement.
Global analysis of time-resolved fluorescence decay
monitored at multiple emission wavelengths reveals
sub-nanosecond lifetime components which dominate
the overall intensity. Based on structure and sequence analysis of several homeodomains, we deduce
that quenching is due to a transient, excited-state
NH . . . ␲ hydrogen bond involving Trp 48 and a conserved aromatic residue at position 8. Additionally,
both time-resolved fluorescence of indole-benzene mixtures and an electrostatic model of the proposed
tryptophan-aromatic interaction substantiate different aspects of this mechanism. A survey of the Protein
Data Bank reveals many proteins with tryptophanaromatic pairs where the indole nitrogen participates
in a NH . . . ␲ hydrogen bond with the ring of another
aromatic residue. Chemical denaturation of one protein found in this survey, human fibronectin type III
module 10, causes an enhancement of the fluorescence quantum yield. This unique interaction has
implications for many other systems and may be
useful for studying larger, multi-tryptophan containing proteins. Proteins 2000;40:112–125.
© 2000 Wiley-Liss, Inc.
Key words: NH . . . ␲ hydrogen bond; fluorescence;
protein unfolding; fibronectin type III;
quenching
INTRODUCTION
Homeotic proteins are transcription factors that control
body patterning of an organism during development. They
contain a ⬃60 amino acid long element, the homeodomain
(HD), which confers DNA binding specificity. One of the
striking features of this class of proteins is the extensive
sequence and structural homology found among diverse,
distantly related members. Structures of Drosophila HDs
such as Antp,1 Ubx,2 Eng,3 the yeast HDs Mata1 and
Mat␣2,4 the murine Msx-1,5 and several other members of
the HD class all show a similar helix-turn-helix fold. All of
these proteins have a conserved tryptophan on the DNA
recognition helix (helix III).6,7 For many of these HDs, this
tryptophan is the only one present, making interpretation
©
2000 WILEY-LISS, INC.
of its fluorescence considerably more straightforward. As a
result, we have an unique opportunity to look at several
similar environments around the buried tryptophan and
potentially make structurally accurate interpretations of
the fluorescence data.
The conserved Trp 48 of HDs from a variety of organims
is characterized by a low fluorescence quantum yield (⌽F).
The single tryptophan RNA-binding Bicoid from Drosophila shows a twenty fold enhancement in signal upon
unfolding by chemical denaturation.8 Mat␣2 from yeast
has two tryptophans, both on the recognition helix: one
buried in the globular core and the other exposed to
solvent. Heating Mat␣2 to the melting temperature of
56°C causes a doubling of intensity and a red-shift in the
peak emission wavelength (Carra and Privalov, unpublished data). Chemical denaturation of both wild-type
Msx-1 from mouse and mutant version of Msx-1, where a
large number of nonconsensus residues are changed to
alanine, causes a significant signal enhancement and a
red-shift of the emission spectrum of the single tryptophan.9 The human HD Pbx-1 which belongs to the TALE
HD class10 contains a single tryptophan with an extremely
low quantum yield and a peak emission of 310 nm.11
Herein, we demonstrate a similar phenomenon in three
Drosophila HDs: Ubx, Eng, and Antp(C39S), where chemical denaturation results in an enhancement of the ⌽F and
a red-shift of peak emission wavelength.57 Direct structural data is available for all of these proteins except
Bicoid and Msx-1, although a homology-based structure
has been constructed for Msx-1.5 One may attribute an
intensity enhancement upon unfolding to specific quenching interactions with polar side chains or backbone groups
in the folded protein.12–14 A closer examination of the
side-chain environment around Trp 48 in these structures
may suggest a mechanism for the low ⌽F in HDs.
The peptide backbone has been shown to quench tryptophan in aqueous solution, but it is not known whether
similar effects are observed inside a protein.12 Studies of
Abbreviations: HD, homeodomain; Ubx, Ultrabithorax; Eng, Engrailed; Antp, Antennapedia; FnIII, fibronectin type III; ⌽F, fluorescence quantum yield; DAS, decay-associated spectra; TRES, timeresolved emission spectra; PDB, protein data bank; GnHCl, guanidine
hydrochloride.
Grant sponsor: National Science Foundation; Grant number: MCB9810812.
*Correspondence to: Vikas Nanda, Department of Biology, Johns
Hopkins University, Baltimore, Maryland 21218. E-mail: vikas@jhu.edu
Received 12 October 1999; Accepted 18 February 2000
113
FLUORESCENCE OF HOMEODOMAINS
specific quenching mechanisms of amino acid functional
groups have shown that lysine and tyrosine quench by
excited-state proton transfer while glutamine, asparagine,
glutamic acid, aspartic acid, cysteine and histidine quench
by excited-state electron transfer.13 Disulfides in the proximity of tryptophan can be a strong quencher.15 However,
these same studies show that most aliphatic residues and
phenylalanine have negligible effects on tryptophan emission at physiological pH. Aromatic and aliphatic residues,
including tryptophan, extensively populate the hydrophobic core of most globular proteins. Due to the absence of
quenching residues, buried tryptophans generally have a
high ⌽F.
The steady state and time-resolved fluorescence of several Drosophila homeodomains in both the native and
denatured state are analyzed and compared for spectroscopic insight into the nature of the side-chain environment around Trp 48. The unusual fluorescence behavior of
the HDs is attributed to a NH . . . ␲ hydrogen bond (also
known as an atypical or unconventional hydrogen bond)
exciplex between the indole nitrogen of Trp 48 and the
center of the aromatic ring of residue 8. This is supplemented with measurements done on indole-benzene mixtures and electrostatic calculations of a tryptophanphenylalanine model interaction. Finally, we survey the
PDB for other proteins with similar interactions in order
to uncover systems where tryptophan may be quenched
due to the formation of such a complex.
MATERIALS AND METHODS
Materials
Ubx HD plasmid was obtained from Tom Tulius and
protein was purified according to the protocol outlined in
Ekker et al.16 Eng protein was obtained from Neil Clarke
and Antp(C39S) was obtained from Peter Privalov. Ubx
and Eng were stored in phosphate buffer (50 mM K2HPO4/
KH2PO4 pH 7.5, 250 mM NaCl, 1 mM dithiothreitol) and
Antp(C39S) was stored in glycine buffer (20 mM glycineHCl pH 5.0, 100 mM NaCl). Human fibronectin Type III
module 10 was provided by Kenneth Ingham.
Steady State Spectroscopy
All steady-state excitation and emission data was measured using a SLM-48000 spectrofluorometer. The excitation and emission slit widths were 4 nm. For emission
spectra, the emission polarizer was vertical and the excitation polarizer was set to the magic angle. Emission spectra
were corrected for instrumental sensitivity using correction factors obtained with a rhodamine quantum counter.
Temperature in the cuvette was maintained at 25°C using
a Neslab temperature controlled water bath. To remove
scattering aggregates, protein was clarified with a low
protein binding 0.22 micron filter (Millipore Corp., Bedford
MA) prior to measurements. Protein concentrations were
kept at ⬃10 ␮M and low light intensities were used to
reduce effects of photobleaching.17 All buffers were measured independently and found to be free of contaminating
fluorescence.
Time-Resolved Fluorescence Spectroscopy
Time-correlated single photon counting measurements
were performed with a picosecond dye laser pumped by a
mode-locked yttrium/aluminum-garnet laser (Spectra
Physics 3000 series) as the light source. Dye laser output
was frequency doubled to 289 or 296 nm to excite tryptophan. Neutral density filters were placed in the excitation
path to attenuate the excitation beam and reduce photobleaching. Emission slits were 1 nm and polarizers were
set to magic angle. The fluorescence emission was monitored at a series of wavelengths between 295 and 450 nm
in 5 nm intervals using a single-photon-counting apparatus with time-resolution of ⬃60 ps. A Ludox scattering
solution was used to measure the instrument response.
The series of decay curves at multiple emission wavelengths were globally fit using the method of least squares
to four discrete exponentials18 with the pre-exponential
factors varying as a function of wavelength (Eq. 1). I is
intensity, ␭em is emission wavelength, t is time, ␣ is the
pre-exponential factor or amplitude and ␶ is the lifetime.
I共t, ␭em兲⫽
冘
␣i共␭em兲exp共⫺t/␶i兲
(1)
i
Wavelength-averaged amplitude components for each lifetime were calculated using the single wavelength amplitudes from the global fit (Eq. 2):
具␣i典⫽
冘
␣i共␭em兲/关
␭em
冘冘
i
␣i共␭em兲兴
(2)
␭em
The amplitude weighted average lifetimes therefore is
simply (Eq. 3):
具␶典⫽
冘
具␣i典␶i
(3)
i
Intensity weighted DAS were generated by plotting the
relative contribution of each lifetime component to the
emission intensity at each of the wavelengths monitored
(Eq. 4).19 –21
Ii共␭em兲⫽␣i共␭em兲␶i
(4)
TRES were generated by fitting the series of emission
intensities monitored at successive wavelengths at fixed
time intervals after excitation.
Model Studies of Indole in Benzene
Steady state and time-resolved measurements of 50 ␮M
indole (Aldrich Chemical Co., Milwaukee, WI, 99⫹% pure)
solutions in cyclohexane (Sigma Chemical Company, St.
Louis, MO) were measured as a function of added benzene
(Sigma). Benzene and cyclohexane were measured under
identical instrument settings to test for background fluorescence contamination, which was not found. We selected
289 nm as the excitation wavelength because it excites into
a major absorption band of indole, and because absorbance
of benzene at 289 is small enough that additions of
benzene to indole in cyclohexane did not cause significant
inner filter artifacts. Emission was monitored at 300 and
310 nm. The bimolecular quenching constant Ksv for the
114
V. NANDA AND L. BRAND
benzene-indole interaction was determined by fitting the
single exponential fit lifetime data to the Stern-Volmer
equation (Eq. 5).22 ␶0 is the lifetime at zero benzene
concentration.
␶0/␶⫽Ksv 关benzene兴⫹1
(5)
1
NR⫽cenar⫺N␧Trp
(9c)
␾⫽acos关共W䡠A兲/共兩W兩兩A兩兲兴
(10a)
2
1
2
1
W⫽共C␧Trp
⫺C␦Trp
兲⫻共C␧Trp
⫺N␧Trp
兲
(10b)
1
1
⫺C␥ar兲⫻共C␦ar
⫺C␨ar兲
A⫽共C␦ar
(10c)
Time-resolved fluorescence decay of indole dissolved in
100% benzene excited at 289 nm was monitored at multiple emission wavelengths between 290 and 400 nm in 5
nm intervals. This data was globally analyzed to generate
DAS and TRES.
Interactions were recorded if the distance between the
indole nitrogen and the center of the ring was less than or
equal to 6.00 Å. The survey includes all structures deposited in the PDB prior to June, 1999.
Hydrogen Bond Energetics
RESULTS
HD Steady State Fluorescence
The potential functions used for modeling the NH . . . ␲
hydrogen bond are the sum of the van der Waals interaction and the electrostatic interaction between the NH and
the atoms of the aromatic group.23 The van der Waals
interaction is modeled by a 12-6 Lennard-Jones function
(Eq. 6).
UvdW⫽ε关共r0/r兲12⫺2共r0/r兲6兴
(6)
Here, r is the distance between the two atoms, r0 is the
distance at the minimum energy of the van der Waals
potential and ε is an energy parameter that depends on
atom type. The pairwise parameter values used are:
r0(HH) ⫽ 2.852 Å, ε(HH) ⫽ 0.038 kcal/mol, r0(CH) ⫽ 3.469
Å, ε(CH) ⫽ 0.038 kcal/mol, r0(NH) ⫽ 3.299 Å, ε(NH) ⫽
0.125 kcal/mol, r0(NC) ⫽ 4.014 Å, ε(NC) ⫽ 0.125 kcal/mol.
The electrostatic interaction is given by a Coulombic term
(Eq. 7).
Uel⫽332䡠qiqj/r
(7)
Partial charges for the indole were taken from semiempirical calculations on 3-methylindole in the ground
and excited-state.24 Charge assignments for the Phe side
chain were taken from studies of Phe–water interactions.25 The total energy is the sum of all pairwise electrostatic and van der Waals interactions.
Structural Survey of the PDB
The C program AROMATIC was designed to examine
PDB26 files for interactions between the indole nitrogen
and the center of an aromatic residue (only Tyr or Phe).
Figure 1 shows the three geometric constraints considered. The following are the calculations used to obtain d, ␪
and ␾ (Eqs. 8 –10 vectors in bold, ‘ar’ subscript denotes
vector for the aromatic acceptor):
1
d⫽兩N␧Trp
⫺cenar兩
(8a)
cenar⫽共C␥ar⫹C␨ar兲/2
(8b)
␪⫽acos关共PN䡠NR兲/共兩PN兩兩NR兩兲兴
(9a)
1
PN⫽P⫺N␧Trp
(9bi)
2
1
⫹C␦Trp
兲/2
P⫽共C␧Trp
(9bii)
共pseudoatom in same direction as Hε1兲
Two single tryptophan-containing HDs, Eng, and Ubx,
show a significant enhancement in intensity upon loss of
native structure. Ubx has a twenty-fold increase in 8.0 M
urea and a 20 nm red-shift in peak emission wavelength
from 335 nm to 355 nm (Fig. 2A). Eng shows a four-fold
enhancement and a similar red-shift upon unfolding (Fig.
2B). Antp(C39S), which contains two tryptophans, shows a
four-fold increase and a 5 nm red-shift with GnHCl
unfolding (Fig. 2C).
HD Time-Resolved Fluorescence
Table I summarizes the global analysis of Ubx, Eng, and
Antp(C39S) fluorescence decay. Tryptophan has a strong
absorption band at 289 nm, making this generally a good
excitation wavelength to use. Additionally, measurements
are done at 295 nm in order to minimize tyrosine fluorescence contamination. The spectroscopic contributions of
tyrosine are small, and do not significantly affect the
lifetime distribution. In the native state, Ubx fluorescence
decay is dominated by two sub-nanosecond lifetime components which account for 82–90% of the excited-state fluorophore population (which is proportional to the preexponential factor, ␣, if the radiative decay rate of
tryptophan is assumed to be constant for all species,
generally a fair assumption for proteins).27 The two longer
lifetimes account for only 10 –18% of the total population,
but contribute significantly to the intensity (which is
proportional to the product of ␣ and ␶). Unfolding of Ubx
alters the lifetime distributions dramatically such that the
dominant lifetime is ⬃3.5 ns. Similar behavior is observed
for both Eng and Antp(C39S).
Intensity-weighted DAS of Eng in the native and denatured state show how the relative contributions of the
different lifetime components change as a function of
wavelength (Fig. 3A). In the native protein, the two
sub-nanosecond lifetimes have blue-shifted associated spectra around 330 nm, while the longer lifetimes have peak
emission around 350 nm. In the denatured protein, none of
the lifetime components have a peak emission shorter
than 350 nm, consistent with the signature of a solventexposed tryptophan in the denatured state. It is obvious
that the two shortest lifetimes account for a significant
fraction of the overall emission intensity in native Eng,
while the 3.20 ns lifetime accounts for the majority of the
fluorescence intensity in unfolded protein. The DAS of Ubx
FLUORESCENCE OF HOMEODOMAINS
115
Fig. 1. Orientation parameters for Trparomatic interactions: Three geometric constraints used to characterize the interaction
between tryptophan and adjacent aromatic side
chains. (A) d, distance, between the indole
nitrogen and the geometric center of the aromatic ring. (B) ␪, angle of projection, between
the direction of the indole nitrogen to its proton
and the indole nitrogen to the ring center. (C) ␾,
interplanar dihedral angle, between the planes
of tryptophan and another aromatic residue.
(data not shown) have similar lifetime distributions as
Eng for both the native and denatured state.
Antp(C39S), with a second exposed tryptophan at position 56, yields different DAS (Fig. 3B). Both native and
unfolded protein DAS are dominated by longer lifetime
species. Still, sub-nanosecond lifetimes contribute more to
native state fluorescence than to unfolded protein fluorescence.
Time-resolved emission spectra (TRES) are effectively
snapshots of the emission spectrum at various times after
excitation. TRES that change over time are indicative of
relaxation of the environment during the lifetime of the
excited-state, or of discrete excited-state heterogeneity, or
of both phenomenon simultaneously. The TRES of Eng
(Fig. 4) are representative of what we also see in
Antp(C39S) and Ubx. The native state TRES show a
significant time dependence for peak emission wavelength. It is difficult to conclude whether this is due to
relaxation or to heterogeneity because the native protein
has a very low ⌽. Even a minute fraction of unfolded
protein could make TRES evolve towards longer wavelengths. The TRES of denatured Eng show little change in
spectral shape with time, which is consistent with a highly
exposed residue, where water relaxes around tryptophan
in times faster than the resolution of the lifetime instrument.
Time-Resolved Fluorescence of Indole-Benzene
Mixtures
It has been demonstrated in the chemical literature that
indole fluorescence is quenched in the presence of benzene.28 Measuring the emission decay kinetics of indole in
cyclohexane as a function of benzene concentration allows
us to determine whether quenching is due to ground or
excited-state interactions. Previous studies describe a
biexponential decay in the presence of benzene, reporting
that benzene emission adds an additional component to
the signal.29 We find that the decay of indole is monoexponential up to 10.0 M benzene in cyclohexane with excellent
␹2 values. Despite this discrepancy, the results from the
two studies are comparable. Our data gives a Ksv of 1.32
and shows that an exciplex is formed between the indole
nitrogen and the benzene ring.29 It should be noted that
the time-resolved emission decay of indole in 11.22 M
(100%) benzene is not monoexponential, and in fact requires four lifetimes to fit the data. This may be due to the
presence of weaker van der Waals interactions of indole
with a second benzene that only occurs at very high
116
V. NANDA AND L. BRAND
Fig. 2. Steady state fluorescence
emission spectra of several HDs: Relative emission spectra (left) and normalized spectra (right) included for (A)
Ubx in storage buffer and storage
buffer ⫹ 8 M urea, (B) Eng in storage
buffer and storage buffer ⫹ 5.4 M
GnHCl, (C) Antp(C39S) in storage
buffer and storage buffer ⫹ 5.4 M
GnHCl. N denotes native protein spectra and D denotes spectra of protein in
denaturant. Excitation wavelength is
295 nm for all of these measurements.
concentrations.30,31 There are some similarities of the
indole-benzene DAS and TRES with those of HDs (Fig. 5).
The dominant emission component in the DAS is a 0.47 ns
lifetime. The TRES evolve during the lifetime of the
excited-state with a broad spectral shift, although the
abruptness of the shift is more indicative of heterogeneity
than relaxation.
Electrostatic Model of the NH . . . ␲ Interaction
Although recent experimental studies have directly
observed the covalent character of the hydrogen bond in
proteins,32,33 the attractive forces that induce a hydrogen bond to form can be well described by electrostatic
modeling of the interaction. Generally, carbon is not a
good hydrogen bond acceptor. However, in an aromatic
ring, the relative geometry of the six partially negative
carbons forms a composite favorable charge that can
participate in a hydrogen bond with a proton donor such
as nitrogen. An atomic charge approximation which
ignores the presence of delocalized electron clouds and
treats benzene as an array of C-H dipoles has been
successfully applied to modeling the NH-benzene interaction, 23 and phenylalanine-phenylalanine interactions.34
Upon excitation of tryptophan, the dipole moment increases substantially and changes direction, which is
reflected by a change in the partial charge distribution on
the atoms of indole.24 Quantum chemical calculations of
the ground and first singlet excited-state show that indole
nitrogen goes from a partial negative charge to a partial
positive charge upon excitation. If tryptophan is involved
in a NH . . . ␲ interaction, making the nitrogen positive
should strengthen the electrostatic interaction with the
carbons of benzene. Figure 6 shows the energy of a
NH . . . ␲ hydrogen bond where the charges on the indole
are those assigned for 3-methylindole in the ground (S0)
117
FLUORESCENCE OF HOMEODOMAINS
TABLE I. Four Exponential Global Fit to Native and Denatured Homeodomains at Two Excitation Wavelengths†
Native
Denatured
289 nm
Ubx
␶
0.11
0.68
2.49
6.01
295 nm
␣
0.69
0.21
0.08
0.02
␶
0.18
0.80
2.72
6.89
具␶典 ⫽ 0.54
␹2 ⫽ 1.261
289 nm
␣
0.55
0.27
0.16
0.02
␶
0.38
1.45
3.67
9.16
具␶典 ⫽ 0.89
␹2 ⫽ 1.292
295 nm
␣
0.20
0.32
0.47
0.01
␶
0.28
1.26
3.57
7.56
具␶典 ⫽ 2.35
␹2 ⫽ 1.053
␣
0.16
0.30
0.52
0.02
具␶典 ⫽ 2.43
␹2 ⫽ 1.089
Eng
289 nm
␶
0.15
0.48
1.95
4.78
具␶典 ⫽ 0.62
␹2 ⫽ 1.042
Antp(C39S)
289 nm
␶
0.14
0.84
2.52
4.63
具␶典 ⫽ 1.66
␹2 ⫽ 1.042
295 nm
␣
0.52
0.36
0.09
0.04
␶
0.17
0.49
2.14
5.07
289 nm
␣
0.53
0.37
0.07
0.03
␶
0.28
1.16
3.00
4.28
具␶典 ⫽ 0.57
␹2 ⫽ 1.030
␶
0.09
0.91
2.83
5.64
␶
0.28
1.16
3.20
4.91
具␶典 ⫽ 2.33
␹2 ⫽ 0.997
295 nm
␣
0.25
0.27
0.39
0.09
295 nm
␣
0.15
0.27
0.43
0.16
具␶典 ⫽ 2.26
␹2 ⫽ 1.007
289 nm
␣
0.44
0.23
0.31
0.03
具␶典 ⫽ 1.30
␹2 ⫽ 1.012
␶
0.29
1.25
3.02
4.61
␣
0.15
0.28
0.50
0.06
295 nm
␣
0.14
0.28
0.47
0.10
具␶典 ⫽ 2.27
␹2 ⫽ 0.998
␶
0.07
0.68
2.30
4.04
␣
0.15
0.21
0.38
0.25
具␶典 ⫽ 2.03
␹2 ⫽ 1.047
Lifetimes are globally fixed across all emission wavelengths. ␣s are summed over all wavelengths and normalized. 具␶典 is ␣ weighted: ¥ ␣i␶i/¥ ␣i 䡠 ␹2
values are for the global analysis.
†
and first singlet excited-state (1La).24 The energy of the
hydrogen bond becomes more favorable in the excitedstate.35 It’s also interesting to note that the equilibrium
distance from the nitrogen to the center of the aromatic
ring decreases from 3.78 Å in the S0 state to 3.65 Å in the
1
La state.
DISCUSSION
The low quantum yield of Trp 48 is a prevalent phenomenon in HDs. The challenge of this study has been to
develop a model of quenching that is consistent with past
and present observations in the context of known structures. The steady-state fluorescence spectra of the three
HDs included in this study and other work done on Mat␣2,
Msx-1, and Bicoid show a red-shift upon unfolding consistent with the transport of Trp 48 from a highly apolar
environment into a polar solvent. The large intensity
increase is observed in each of the HDs we have surveyed,
suggesting that some common native structure interaction
quenches Trp 48. The magnitude of the intensity change
varies for different HDs. In summary, Bicoid8 and Ubx
show the largest change (⬃20 fold), while Msx-19 and Eng
show a 4 –5 fold enhancement. Antp(C39S) shows a similar
change, and the increase attributed to Trp 48 is believed to
be the dominant one. Trp 56 of Antp(C39S) is more
exposed and should only increase slightly in the presence
of denaturant.* The doubling of intensity observed during
Mat␣2 melting may understate the enhancement of Trp 48
for two reasons: first, Mat␣2 also has two tryptophans in
similar environments to Antp so the change in Trp 48 may
be partially masked by Trp 56, and second, tryptophan
fluorescence decreases with increasing temperature, so
presumably, chemical denaturation of Mat␣2 would show
an even greater change in signal intensity. By looking at
the details of the side-chain environment in many of these
homeodomains, we can attempt to infer what type of
interactions are responsible for reducing the Trp 48 ⌽F.
Structural Analysis of HDs
The crystal and NMR structures of Antp, Eng, Ubx, and
Mat␣2 are scrutinized for details on the side-chain environment of Trp 48. Any heavy atoms within 6.00 Å of the
tryptophan Cε2 as determined using the NEIGHBORS
function of Insight 95.036 are considered for possible
interaction with the indole fluorophore (Fig. 7). Due to the
high degree of sequence and structural homology between
HDs, residues 8, 13, 16, 17, 49, and 52 are always seen in
contact with Trp 48. Which of these interactions could give
rise to the characteristic low ⌽F of HDs? Most contacts
*Increasing [urea] or [GnHCl] generally causes a small enhancement and no detectable spectral shift when unaccompanied by a
structural change.
118
V. NANDA AND L. BRAND
Fig. 3. Intensity weighted DAS of
HDs excited at 295 nm using four
global lifetimes: (A) Native (left) and
denatured (right) Eng. Lifetime values
in Table I. (B) Native (left) and denatured (right) Antp(C39S). Lifetime values in Table I.
Fig. 4. TRES of Eng excited at 289
nm: in the native state (left) and denatured state (right). Emission spectra at
six time points are shown: 0, 0.1, 0.3,
1.0, 3.0, and 10.0 ns after excitation.
Spectra evolve towards longer wavelengths with increasing time.
observed are with aromatic ring groups, aliphatic sidechains, or the alkyl chains of polar residues. The exception
is in the crystal structure of Eng where several terminal
amines are found in proximity to Trp 48. N␩1 and N␩2 of
Arg 5 and the N␨ of Lys 52 and Lys 55 are potentially very
good quenchers of tryptophan fluorescence. Additionally,
bound water found in the HD/DNA interface may also
reduce the Trp 48 ⌽F. However, neither bound water nor
polar side-chain amine interactions are consistently found
for all the structures investigated and therefore would not
provide a general explanation of quenching in HDs. Additionally, quenching by solvent is not sufficient to account
for the magnitude of the quantum yield reduction.37 We
therefore suggest that the primary mechanism of quenching is by interactions with the aromatic side-chains of
adjacent phenylalanines or tyrosines.
The four aromatic residues in the core of Eng (Phe 8,
Phe 20, Trp 48, and Phe 49) pack in a “herringbone”
FLUORESCENCE OF HOMEODOMAINS
119
Fig. 5. Indole in 100% benzene
excited at 289 nm using four global
lifetimes: (A) Intensity weighted DAS:
␶1 ⫽ 0.47 ns, ␶2 ⫽ 2.98 ns, ␶3 ⫽ 1.67
ns, ␶4 ⫽ 11.51 ns. (B) TRES: Emission
spectra at six time points are shown: 0,
0.1, 0.3, 1.0, 3.0, and 10.0 ns after
excitation. Spectra evolve towards
longer wavelengths with increasing
time.
Fig. 6. Energy of a Trp-Phe NH . . . ␲ hydrogen bond: Interaction
energy as a function of the distance between the indole nitrogen and the
center of the aromatic ring.
conformation.3 Of these, residue 8 is either a phenylalanine or a tyrosine for the HDs included in this study, and
is aromatic in 80% of known HDs.6 Residue 49 is always
phenylalanine except in the case of Mat␣2, where it is a
valine. Residue 20 is always phenylalanine, but does not
make contact with Trp 48. Tyrosine has been found to
quench tryptophan through excited-state proton transfer,13 but the same study finds that phenylalanine
generally does not affect tryptophan ⌽ F . If a universal
mechanism for the ⌽ F of HDs does in fact exist, it
presumably involves residue 8, which is always in
contact with Trp 48 and is always aromatic. Additionally, this mechanism must explain the interchangeability of phenylalanine and tyrosine at position 8. Evidence
for just such a mechanism consistent with both fluorescence and structural data comes from another type of
protein which also has a low ⌽ F .
Steady state measurements of the immunophilin
FKBP12 find Trp 59 has a ⌽F of 0.014,38 which is a factor
of ten lower than free tryptophan.39 Urea-induced unfolding of human FK506 binding protein shows a 15-fold
enhancement.40 NMR characterization of the FKBP59
immunophilin, which is homologous to FKBP12, demonstrates the presence of a hydrogen bond between the indole
nitrogen of Trp 89 and the center of the aromatic ring of
Phe 129.41 The two rings are oriented perpendicularly,
with the indole nitrogen proton pointing directly towards
the center of the phenylalanine ring. Time-resolved fluorescence measurements on FKBP12 further suggests that the
presence of this hydrogen bond correlates with the weak
fluorescence of Trp 59.42 We suggest that a similar interaction between Trp 48 and residue 8 may be responsible for
the low ⌽F observed in HDs.
The hypothesis that fluorescence is quenched by an
atypical hydrogen bond from Trp 48 to residue 8 is
predominantly based on elimination of other known
mechanisms of tryptophan quenching. Assuming that
the mechanism is a general one for the entire class of
proteins, driven by a common interaction, we can eliminate quenching by polar residues, cysteine, histidine,
water, or backbone amides. However, if a tryptophanaromatic pair is indeed responsible for Trp 48 low ⌽ F ,
then why do X-Ray and NMR-derived structures not
show clear evidence of an NH . . . ␲ interaction? The
majority of structures show Trp 48 in an orientation
where the C␦ 1 is pointing to the ring instead of the
nitrogen. It could be argued that most of the structures
available are DNA/protein co-crystals, which influences
the tryptophan orientation. This would imply that adding specific DNA to a HD should cause an increase in
intensity similar to protein unfolding, but we have not
been able to observe this. Additionally, structures of
protein in the absence of DNA have moderately improved tryptophan geometries for hydrogen bond formation, but these still diverge significantly from tryptophan–aromatic interactions such as those seen in
immunophilins. Therefore, DNA binding-induced structural changes do not seem to be an issue.
One possibility is that excitation of Trp 48 facilitates an
excited-state rearrangement, allowing hydrogen bond for-
Fig. 7. Side chain environment around Trp 48: (A) Eng–1ENH, (B) Antp/DNA–9ANT, (C) Ubx/Exd/DNA–1B8I and (D) Mat␣2/Mata1/DNA–1AKH. Trp 48 and residue 8 (Trp 179 and Phe 136 in
1AKH) are highlighted. Individual crosshatches represent coordinates of bound water oxygen atoms. Graphics generated using Insight 95.0.34
FLUORESCENCE OF HOMEODOMAINS
121
Fig. 8. Excited state rearrangement model: This cartoon depicts the
rotation believed to be required for formation of the hydrogen bond in
HDs. Through graphical analysis of available structures, a 70 – 80°
rotation in the direction shown is sufficient to bring the indole nitrogen in
proximity to the aromatic acceptor. The rotation along the C␣-C␤ bond
may either be a result of picosecond fluctuations in structure or directed
electrostatically driven motion of the side chain in the indole plane as a
result of its change in dipole moment.
mation and quenching. An in-plane rotation of the indole
side chain would allow the necessary interactions to occur
(Fig. 8). There are clues in the fluorescence behavior that
substantiate this mechanism.
tophans undergoing an excited-state rotation and hydrogen
bond formation as indicated in the cartoon (Fig. 8).
There are two possible reasons for an excited-state
rearrangement to occur. It is known that local side-chain
fluctuations occur on the picosecond time-scale.43 It may
be that Trp 48 fluctuates rotationally in its plane during
the lifetime of the excited-state. If one of these motions
brings the indole NH close enough to the aromatic acceptor, our electrostatic model suggests that there will be a
strong attraction between the residues resulting in hydrogen bond formation and subsequent de-excitation. The
second mechanism postulated is an induced excited-state
rearrangement based on the change in tryptophan dipole
moment upon excitation. The dipole moment of the indole
ring changes magnitude from 2.1 to 5 D upon excitation
and changes in direction.44 This change in dipole moment
may induce torque on the indole side chain due to the local
electric field which is relaxed by the in-plane rotation.
Such a directed process would make quenching more
efficient and potentially account for the low ⌽F observed in
HDs. This would also explain TRES of HDs in the native
state, where the rotational relaxation of transition energy
(E 䡠 ⌬␮) is reflected in a red-shifting emission maximum
with time.
The indole-benzene complex behaves as a pure dynamic
quenching system where both 具␶典 and ⌽F decrease six fold
in the presence of benzene. The low Ksv of benzene
quenching of indole suggests that the efficiency of this
interaction is low. This argues strongly for quenching
caused only by collisions where both indole and benzene
need to be in a particular relative orientation. All other
things being equal, quenchers with spherical symmetry
such as iodide would therefore be relatively more efficient
since there would be fewer orientation constraints on the
quenching interaction. The bimodal nature of the indole-
Excited State Rearrangement and Quenching
The time-resolved fluorescence behavior of HDs brings
up a number of issues. One notable discrepancy is that for
Ubx and Antp(C39S):
␶native
⌽native
⫽
␶denatured ⌽denatured
(11)
This is often indicative of a mixture of static and dynamic
quenching mechanisms.22 If the ratios are equal, the mechanism can be considered purely dynamic, and if ␶native ⫽
␶denatured then the mechanism can be described as purely
static. However, the decay of both native and denatured
homeodomains are multi-exponential, making it difficult to
assign meaningful values for ␶native and ␶denatured. One may
summarize the lifetime parameters by describing each state
with an average lifetime 具␶典, but this can be misleading. The
intensity weighted 具␶典 will depend strongly on tiny fractional
contributions from longer lifetimes. A longer lifetime from a
minute fraction of unfolded or misfolded protein in a native
state ensemble could easily increase the 具␶典 significantly. The
␣ weighted 具␶典 will depend strongly on the resolution of the
instrument, and the fitted pre-exponential factors for short
lifetimes may be inaccurate. Assuming the time resolution of
our instrument is adequate to resolve amplitudes for the
short lifetime components, it suggests that Trp 48 is involved
in both ground and excited-state quenching processes. The
ground-state component may be a fraction of tryptophan
already forming an atypical hydrogen bond with residue 8
and the excited-state component would represent those tryp-
122
V. NANDA AND L. BRAND
TABLE II. Several Proteins with Structural Evidence for NH . . . ␲ Hydrogen Bonds†
PDB id
Protein
Structure type
Residues
dÅ
␪°
␾°
1lqi
1a88
1axi
1xtc
1epj
1fgp
1iln
1lis
1mfn
1pls
1waj
3cp4
1rlr
1vrt
1igt
1pcr
1oya
2puc
1luc
2fke
1hom
9ant
1b8i
1akh
1enh
3hdd
insecticidal ␣ scorpion toxin
chloroperoxidase L
HGH and receptor
cholera toxin
epidermal growth factor
phage coat protein
interleukin-2
red abalone lysin
mouse fibronectin
pleckstrin homology domain
bacteriophage DNA polymerase
cytochrome P450CAM
ribonucleotide reductase R1
HIV-1 reverse transcriptase
immunoglobulin
photosynthetic reaction center
old yellow enzyme
purine repressor PurR w/DNA
bacterial luciferase
human FKBP12
Antp
Antp/DNA
Ubx/Exd/DNA
Mat␣2/Mata1/DNA
Eng
Eng/DNA
NMR/Md11
Xray/1.9 Å
Xray/2.1 Å
Xray/2.4 Å
NMR/Md 4
NMR/Md 3
theoretical
Xray/1.9 Å
NMR/Md 3
NMR/Md 3
Xray/2.8 Å
Xray/2.3 Å
Xray/2.5 Å
Xray/2.2 Å
Xray/2.8 Å
Xray/2.7 Å
Xray/2.0 Å
Xray/2.6 Å
Xray/1.5 Å
Xray/1.7 Å
NMR/Md 4
Xray/2.4 Å
Xray/2.4 Å
Xray/2.5 Å
Xray/2.1 Å
Xray/2.8 Å
W39-F18
W183-F165
W157-Y174
W88-Y12
W49-Y37
W37-Y32
W121-F117
W62-Y133
W22-Y32
W21-F62
W205-F113
W374-F381
W309-F368
W229-Y183
W47-Y35
W66-Y177
W116-Y196
W147-Y126
W194-Y42
W59-F99
W48-Y8
W48-Y8
W48-Y8
W179-F136
W48-F8
W48-F8
5.86
3.10
3.46
5.53
3.79
4.59
3.14
3.68
3.98
3.61
5.76
3.81
3.43
3.55
4.06
3.79
3.46
2.99
5.65
3.34
4.75
5.65
5.69
5.38
5.21
5.21
22
18
12
15
10
18
10
8
18
18
16
20
20
16
9
14
18
18
18
8
105
85
87
90
85
95
128
106
110
88
88
93
90
94
101
106
105
86
84
95
72
102
83
84
71
90
100
34
21
34
113
88
†
X-ray crystal structures include the reported resolution under Structure Type. NMR structures are referred to by specific
model number. Geometric parameters listed for four homeodomains. Information for both free and DNA bound forms
included for Antp (9ANT, 1HOM) and Eng (3HDD, 1ENH).
benzene TRES may be best described as a mixture of
states: one being the atypical hydrogen bond, and the other
a less specific, stacked van der Waals complex. MassAnalyzed Pulsed Field Threshold Ionization Spectroscopy
measurements of 1:1 indole-benzene clusters in the gas
phase show that the atypical hydrogen bond is the primary
conformation.31
As an aside, it is interesting to note that the lifetime
distributions of unfolded Antp(C39S) and Eng are different are very different (Fig. 3), indicating that the environments around Trp 48 and Trp 56 in the Antp(C39S)
random coil are dissimilar. In Antp(C39S), Trp 48 is
flanked by Ile 47 and Phe 49 while Trp 56 is between Lys
55 and Lys 57. In Eng, Trp 48 is flanked by Ile 47 and Phe
49. If one assumes the denatured state has no persistent
structure, this observation suggests that the difference in
denatured protein DAS signatures is due to possible
electrostatic effects from the presence of the polar lysine
side chains adjacent to Trp 56 of Antp(C39S).
Surveying the PDB
With the evidence mounting for an aromatic mediated
tryptophan quenching in HDs, the question arises as to the
generality of this phenomenon. By surveying the PDB for
proteins with tryptophan-aromatic interactions, we compile a list of candidates that may potentially have a low
tryptophan ⌽F. Three geometric parameters: distance (d),
angle of projection (␪) and dihedral angle (␾) for interact-
ing pairs of tryptophan and phenylalanine or tyrosine are
measured for structures in the PDB (Fig. 1). Potentially
interesting interactions would also include tryptophanaromatic histidine or tryptophan-tryptophan complexes,
but these two cases are excluded in the present study. Only
structures with orientations within 20° of ␾ ⫽ 90° and ␪ ⫽
0° are kept. Table II lists several of the proteins obtained
from this survey. In addition to the immunoglobulin
structure, 1IGT mentioned in Table II, over twenty other
structures of FAB fragments bound to various epitopes are
also culled from the PDB. A large number of immunophilin
mutants, homologues, protein-ligand complexes, etc., are
also found in this survey. 2FKE is included in Table II as a
representative of the interaction geometry in immunophilins. Several of these proteins have a low ⌽F and clearly
show an enhancement upon denaturation. The same geometric parameters are listed for several HD structures. As
previously mentioned, there is no direct observation of the
hydrogen bond in the HD structures, which is reflected in
the values for d, ␾, and ␪.
IL-2 has a tryptophan-phenylalanine pair involved in a
helical i, i ⫹ 4 interaction buried in the hydrophobic core.
The only potential quenching group is a cysteine which is
greater than 6.0 Å away from Trp 121.45 As Table II shows,
the distance and orientation parameters are ideal for
formation of an NH . . . ␲ hydrogen bond between Trp 121
and Phe 117. A crystal structure of human IL-2 (3INK)
shows that the cysteine and a nearby histidine are not in
123
FLUORESCENCE OF HOMEODOMAINS
Fig. 9. Denaturation of human FnIII-10 in GnHCl: Intensity change
(solid line, crosses) is ⬃4 fold. The peak shift represented as a function of
I350nm/I320nm (dashed line, open circles).
bonds have roles in both protein structure and ligand
binding interactions.50 –52 Mutational studies on binding
site residues of the CAP/DNA complex show that introducing an aromatic residue which acts as a H-bond acceptor
can contribute ⬃0.5 to 1 kcal/mol to binding.53 If the
energy of the NH . . . ␲ hydrogen bond is near ⫺2.81
kcal/mol, as the physical model suggests, this would be
significant enough to contribute to the energy of protein
stabilization which is on the order of 5 to 10 kcal/mol.54 It
is a more favorable interaction than typical nonbonding
aromatic-aromatic interactions which are on the order of
1–2 kcal/mol.55 The omnipresence of specific tryptophans
in FnIII and HDs and the conserved adjacent aromatics
suggest that these edge-face interactions are significant to
stability. It is interesting to note that in a sequence
alignment of FnIII domains, the otherwise conserved
Trp-aromatic interaction is absent in interleukin-7 receptor, but it is replaced by a histidine-phenylalanine pair,56
an interaction which has also been implicated in forming
NH . . . ␲ hydrogen bonds in proteins.50 One might speculate that this interaction is conserved not only because of
the energetic contribution, but also due to the specificity of
packing that such an interaction entails. Orienting bulky
aromatics in such a fashion may provide a scaffold for the
remaining core.
CONCLUSIONS
the correct orientation, or in the vicinity to quench Trp
121. Chemical denaturation studies performed on wildtype human IL-2 shows a 50% increase in signal intensity
upon unfolding and a large red-shift of the emission
wavelength peak from 324 nm to ⬃350 nm indicating a
removal of Trp 121 from a hydrophobic, but quenching
environment.46 In the same study, a mutant with an
additional exposed tryptophan also shows an enhancement and a red-shift of emission upon denaturation.
Human FnIII Module 10 has a single tryptophan and an
extremely low ⌽F in the native state. Addition of GnHCl to
native human FnIII module 10 shows a four-fold enhancement and a significant red-shift in emission (Fig. 9). The
emission spectrum of the native protein is dominated by
tyrosine contributions. The same effect has been noted in
studies of human FnIII Module 1.47 There is little structural evidence for other mechanisms of quenching. It is
plausible that the atypical hydrogen bond between Trp 22
and Phe 32 in FnIII modules is responsible for tryptophan
quenching. This mechanism is consistent with sequence
conservation of interacting residues which would give a
general description of the low ⌽F in fibronectins.
Stability and Conservation
The presence of stabilizing edge-face aromatic interactions may be the reason that Trp 22 is highly conserved in
FnIII type folds and why Trp 48 is conserved in HDs. It is
known from experimental studies and physical models
that the energetics of the NH . . . ␲ bond is approximately
half the energy of standard hydrogen bonds found in
proteins.23,48 Generally, these hydrogen bonds lose in
competition to more favorable side chain interactions.49
Still, numerous examples exist where NH . . . ␲ hydrogen
The unusual fluorescence of Trp 48 in HDs cannot be
satisfactorily explained by conventional quenching mechanisms. Structure and sequence analysis indicates that
interactions of Trp 48 with adjacent aromatics may result
in a low ⌽F. The mechanism presented at our current level
of understanding is a transient NH . . . ␲ hydrogen bond
between Trp 48 and the aromatic ring of residue 8 caused
by an excited-state in plane rotation of the indole side
chain. Several other proteins with direct structural evidence of an NH . . . ␲ hydrogen bond have a low ⌽F. A
practical application of this phenomenon to many of the
proteins found in the PDB survey is the potential for using
tryptophan-aromatic pairs to study systems with multiple
tryptophans. As was observed in the case of Antp(C39S),
the sensitivity of total tryptophan emission to conformation is dominated by the enhancement observed in Trp 48.
Even with proteins with a large number of tryptophans, a
conformational change which can effectively switch a
tryptophan “on or off” may cause an interpretable change
in fluorescence. With the accessibility and advantages of
using fluorescence to study conformation and interactions,
knowledge that an atypical hydrogen bond involving tryptophan occurs in a particular system can be very useful.
ACKNOWLEDGMENTS
This work was only possible with the valuable input and
expertise and the materials provided by several people. We
thank Richard Frazee and Tom Tulius for providing the
Ubx plasmid and teaching us the purification, Neil Clarke
for the generous gift of Eng protein and valuable discussions on HD structure, Peter Privalov for providing Antp
protein and communicating valuable information on Mat␣2
124
V. NANDA AND L. BRAND
fluorescence, Zhenlan Li for additional help with the Antp
system, Ken Ingham for the generous gift of human
fibronectin, George Rose, Thomas Woolf, Bertrand GarciaMoreno, Hirsh Nanda, and Alan Grossfield for valuable
discussions on working with the PDB and modeling the
tryptophan-phenylalanine interaction, Dima Toptygin for
training on the fluorescence instrumentation and valuable
discussion on data analysis, Andy Russo and Michael
Rodgers for valuable discussions on pertinent biochemical
techniques and protein purification.
REFERENCES
1. Qian Y, Billeter M, Otting G, Müller M, Gehring W, Wüthrich K.
The structure of the Antennapedia homeodomain determined by
NMR spectroscopy in solution: Comparison with prokaryotic
repressors. Cell 1989;59:573–580.
2. Passner JM, Ryoo HD, Shen L, Mann RS, Aggarwal AK. Structure
of a DNA-bound Ultrabithorax-Extradenticle homeodomain complex. Nature 1999;397:714 –719.
3. Clarke ND, Kissinger CR, Desjarlais J, Gilliland GL, Pabo CO.
Structural studies of the engrailed homeodomain. Protein Sci
1994;3:1779 –1787.
4. Li T, Stark MR, Johnson AD, Wolberger C. Crystal structure of the
MATa1/MAT␣2 homeodomain heterodimer bound to DNA. Science 1995;270:262–269.
5. Li H, Tejero R, Monleon D, et al. Homology modeling using
simulated annealing of restrained molecular dynamics and conformational search calculations with CONGEN: application in predicting the three-dimensional structure of murine homeodomain
MSX-1. Protein Sci 1997;6:956 –970.
6. Laughon A. DNA binding specificity of homeodomains. Biochemistry 1991;30:11358 –11367.
7. Gehring WJ, Affolter M, Bürglin T. Homeodomain proteins. Annu
Rev Biochem 1994;63:487–526.
8. Subramaniam V, Rivera-Pomar R, Jovin TM. Su-Pos447: the
conserved tryptophan in the recognition helix of the bicoid homeodomain is a sensitive probe of structure and folding. Biophys J
1999;76:A108.
9. Shang Z, Issac VE, Li H, et al. Design of a “minimAL” homeodomain: the N-terminal arm modulates DNA binding affinity and
stabilizes homeodomain structure. Proc Natl Acad Sci 1994;94:
8373– 8377.
10. Sanchez M, Jennings PA, Murre C. Conformational changes
induced in Hoxb-8/Pbx-1 heterodimers in solution and upon
interaction with specific DNA. Mol Cell Bio 1997;17:5369 –5376.
11. Bürglin TR. Analysis of TALE superclass homeobox genes (MEIS,
PBC, KNOX, Irogquois, TGIF) reveals a novel domain conserved
between plants and animals. Nucl Acids Res 1997;25:4173– 4180.
12. Chen Y, Liu B, Yu H-T, Barkley MD. The peptide bond quenches
indole fluorescence. J Am Chem Soc 1996;118:9271–9278.
13. Chen Y, Barkley MD. Towards understanding tryptophan fluorescence in proteins. Biochemistry 1998;37:9976 –9982.
14. Harris DL, Hudson BS. Photophysics of tryptophan in bacteriophage T4 lysozyme. Biochemistry 1990;29:5276 –5285.
15. Cowgill RW. Fluorescence and the structure of proteins. XVIII.
Spatial requirements for quenching by disulfide groups. Biochim
Biophys Acta 1970;207:556 –559.
16. Ekker SC, von Kessler DP, Beachy PA. Differential DNA sequence
recognition is a determinant of specificity in homeotic gene action.
EMBO J 1992;11:4059 – 4072.
17. Toptygin D, Rodgers ME, Brand L. Tu-Pos490: Non-exponential
fluorescence decay of photobleached indole and tryptophan. Biophys J 1999;76:A361.
18. Grinvald A, Steinberg IZ. On the analysis of fluorescence decay
kinetics by the method of least-squares. Anal Biochem 1974;59:
583–598.
19. Donzel B, Gauduchon P, Wahl P. Study of the conformation in the
excited-state of two tryptophanyl diketopiperazines. J Am Chem
Soc 1974;96:801– 808.
20. Knutson JR, Walbridge DG, Brand L. Decay-associated fluorescence spectra and the heterogeneous emission of alcohol dehydrogenase. Biochemistry 1982;21:4671– 4679.
21. Ross JBA, Schmidt CJ, Brand L. Time-resolved fluorescence of the
two tryptophans in horse liver alcohol dehydrogenase. Biochemistry 1981;20:4369 – 4377.
22. Lakowicz JR. Principles of fluorescence spectroscopy. New York:
Plenum Press; 1983.
23. Levitt M, Perutz MF. Aromatic rings act as hydrogen bond
acceptors. J Mol Biol 1988;201:751–754.
24. Muiño PL, Harris D, Berryhill J, Hudson B, Callis PR. Simulation
of solvent dynamics effects on the fluorescence of 3-methylindole
in water. SPIE—Time-resolved laser spectroscopy in biochemistry
III 1992;1640:240 –251.
25. Bolis G, Clementi E. Analytical potentials from “ab initio” computations for the interaction between biomolecules. 3. Reliability and
transferability of the pair potentials. J Am Chem Soc 1977;99:
5550 –5557.
26. Bernstein FC, Koetzle TF, Williams GJB, et al. The protein data
bank: a computer-based archival file for macromolecular structure. J Mol Biol 1977;112:535–542.
27. Strickler SJ, Berg RA. Relationship between absorption intensity
and fluorescence lifetime of molecules. J Chem Phys 1962;37:814 –
822.
28. Van Duuren BL. Solvent effects in the fluorescence of indole and
substituted indoles. J Org Chem 1961;26:2954 –2960.
29. Suwaiyan A, Klein KA. Picosecond study of solute-solvent interaction of the excited-state of indole. Chem Phys Lett 1989;159:244 –
250.
30. Hager J, Ivanco M, Smith MA, Wallace SC. Two-color threshold
photoionization spectroscopy of jet-cooled indole clusters. Chem
Phys 1986;105:397– 416.
31. Braun JE, Grebner ThL, Neusser HJ. van der Waals versus
hydrogen-bonding in complexes of indole with argon, water and
benzene by mass-analyzed pulsed field threshold ionization. J
Phys Chem 1998;102:3273–3278.
32. Isaacs ED, Shukla A, Platzman PM, Hamann DR, Barbiellini B,
Tulk CA. Covalency of the hydrogen bond in ice: a direct x-ray
measurement. Phys Rev Lett 1999;82:600 – 603.
33. Wang Y-X, Jacob J, Cordier F, Wingfield P, Stahl SJ, Lee-Huang
S, Torchia D, Grzesiek S, Bax A. Measurements of 3hJNC⬘ connectivities across hydrogen bonds in a 30 kDa protein. J Biomol NMR
1999;14:181–184.
34. Hunter CA, Singh J, Thornton JM. ␲-␲ interactions: the geometry
and energetics of phenylalanine-phenylalanine interactions in
proteins. J Mol Biol 1991;218:837– 846.
35. Pimentel GC. Hydrogen bonding and electronic transitions: the
role of the Franck-Condon principle. J Am Chem Soc 1957;79:3323–
3326.
36. Insight95.0. San Diego: MSI/Biosym; 1995.
37. Gallay J, Vekshin N, Sopkova J, Vincent M. Evidences for
picosecond excited state reactions of indole derivatives in alcoholic
solvents, reverse micelles and proteins. SPIE—time-resolved laser spectroscopy in biochemistry III 1994;2137:390 –399.
38. Silva ND, Prendergast FG. Tryptophan dynamics of the FK506
binding protein: time-resolved fluorescence and simulations. Biophys J 1996;70:1122–1137.
39. Chen RF. Fluorescence quantum yields of tryptophan and tyrosine. Anal Lett 1967;1:35– 42.
40. Egan DA, Logan TM, Liang H, Mtayoshi E, Fesik SW, Holzman
TF. Equilibrium denaturation of recombinant Human FK Binding
Protein in Urea. Biochemistry 1993;32:1920 –1927.
41. Craescu CT, Rouvière N, Popescu A, Cerpolini E, Lebeau M-C,
Baulieu E-E, Mispelter J. Three-dimensional structure of the
immunophilin-like domain of FKBP59 in solution. Biochemistry
1996;34:11045–11052.
42. Rouvière N, Vincent M, Craescu CT, Gallay J. Immunosupressor
binding to the immunophilin FKBP59 affects the local structure
dynamics of a surface ␤-strand: time-resolved fluorescence study.
Biochemistry 1997;36:7339 –7352.
43. McCammon JA, Wolynes PG, Karplus M. Picosecond dynamics of
tyrosine side chains in proteins. Biochemistry 1979;18:927–942.
44. Pierce DW, Boxer SG. Stark effect spectroscopy of tryptophan.
Biophys J 1995;68:1583–1591.
45. Dryden D, Weir MP. Evidence for an acid-induced molten-globule
state in interleukin-2; a fluorescence and circular dichroism study.
Biochim Biophys Acta 1991;1078:94 –100.
46. Weir MP, Chaplin MA, Wallace DM, Dykes CW, Hobden AN.
Structure-activity relationships of recombinant human interleukin 2. Biochemistry 1988;27:6883– 6892.
FLUORESCENCE OF HOMEODOMAINS
47. Litvinovich SV, Novokhatny VV, Brew SA, Ingham KC. Reversible unfolding of an isolated heparin and DNA binding fragment,
the first type III module from fibronectin. Biochim Biophys Acta
1992;1119:57– 62.
48. Knee JL, Khundkar LR, Zewail AH. Picosecond photofragment
spectroscopy. III. Vibrational predissociation of van der Waal’s
clusters. J Chem Phys 1987;87:115–127.
49. Mitchell JBO, Nandi CL, McDonald IK, Thornton JM, Price SL.
Amino/aromatic interactions in proteins: is the evidence stacked
against hydrogen bonding? J Mol Biol 1994;239:315–331.
50. Armstrong KM, Fairman R, Baldwin RL. The (i, i ⫹ 4) Phe-His
interaction studied in a alanine-based ␣-helix. J Mol Biol 1993;230:
284 –291.
51. Perutz MF. The role of aromatic rings as hydrogen-bond acceptors
in molecular recognition. Phi Trans R Soc A 1993;345:105–112.
125
52. Burley SK, Petsko GA. Amino-aromatic interactions in proteins.
FEBS Lett 1986;203:139 –143.
53. Parkinson G, Gunasekera A, Vojtechovsky J, Zhang X, Kunkel
TA, Berman H, Ebright RH. Aromatic hydrogen bond in sequence
specific DNA recognition. Nat Struct Biol 1996;3:837– 841.
54. Pace NC. Conformational stability of globular proteins. Trends
Biochem Sci 1990;15:14 –17.
55. Burley SK, Petsko GA. Aromatic-aromatic interaction: a mechanism of protein structure stabilization. Science 1985;229:23–28.
56. Bazan JF. Structural design and molecular evolution of a cytokine
receptor subfamily. Proc Natl Acad Sci 1990;87:6934 – 6938.
57. Nanda V, Brand L. WPos277 Fluorescence characterization of
homeodomains: Quenching of a buried tryptophan by aromatic
hydrogen bonding associations. Biophys J 1999;76:A448.
Документ
Категория
Без категории
Просмотров
2
Размер файла
303 Кб
Теги
226
1/--страниц
Пожаловаться на содержимое документа