From the Laboratoire de Résonance
Magnétique Nucléaire en Biologie et Médecine
(Unité Propre de Recherche de l'Enseignement Supérieur EA
2230), Faculté de Médecine, CS 34317, Rennes 35043 cedex,
the ¶ Groupe de Recherche en Thérapeutique
anticancéreuse, CNRS FRE 2261, Faculté de Médecine,
CS 34317, Rennes 35043 cedex, the
Laboratoire de Chimie
Organométallique et Biologique, Unité Mixte de Recherche
CNRS 6509, Rennes 35042 cedex, and the ** Laboratoire pour
l'Utilisation du Rayonnement Electromagnétique, Unité
Mixte de Recherche CNRS 130, Centre Universitaire Paris Sud, BP 34,
Orsay 91898, France
Received for publication, July 22, 2002, and in revised form, December 10, 2002
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ABSTRACT |
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Dystrophin is assumed to act via the central rod
domain as a flexible linker between the amino-terminal actin binding
domain and carboxyl-terminal proteins associated with the membrane. The rod domain is made up of 24 spectrin-like repeats and has been shown to
modify the physical properties of lipid membranes. The nature of this
association still remains unclear. Tryptophan residues tend to cluster
at or near to the water-lipid interface of the membrane. To assess
dystrophin rod domain-membrane interactions, tryptophan residues
properties of two recombinant proteins of the rod domain were examined
by 1H NMR and fluorescence techniques in the presence
of membrane lipids. F114 (residues 439-553) is a partly folded protein
as inferred from 1H NMR, tryptophan fluorescence emission
intensity, and the excited state lifetime. By contrast, F125 (residues
439-564) is a folded compact protein. Tryptophan fluorescence
quenching shows that both proteins are characterized by structural
fluctuations with their tryptophan residues only slightly buried from
the surface. In the presence of negatively charged small vesicles, the
fluorescence characteristics of F125 change dramatically, indicating
that tryptophan residues are in a more hydrophobic environment.
Interestingly, these modifications are not observed with F114.
Fluorescence quenching experiments confirm that tryptophan residues are
shielded from the solvent in the complex F125 lipids by a close contact
with lipids. The use of membrane-bound quenchers allowed us to conclude that dystrophin rod domain lies along the membrane surface and may be
involved in a structural array comprising membrane and cytoskeletal
proteins as well as membrane lipids.
Dystrophin is a filamentous 427-kDa protein whose deficiency is at
the origin of Duchenne muscular dystrophy (1). The dystrophin molecule
is composed of four domains: an actin-binding amino-terminal domain, a
rod domain comprising 24 spectrin-like repeats, and a carboxyl-terminal
end made up of cysteine-rich and dystroglycan-interacting domains
anchoring the dystrophin molecule at the sarcolemma (2-4). The
amino-terminal end (5) and a cluster of basic repeats of the rod domain
(6) are clearly associated with F-actin, suggesting a lateral
association between dystrophin and actin. All these associations point
to a model of dystrophin acting as a flexible linker between the
cytoskeleton and the extracellular matrix (7-9). However, it has also
been observed that a repeat of the rod domain is able to modify the
physical properties of lipid membranes containing anionic phospholipids
(10). This result is consistent with the observation that, in the
muscle of patients expressing genetic variants of dystrophin lacking
the carboxyl-terminal end, the truncated dystrophin is nevertheless
well localized at the sarcolemma (11-13). It is also compatible with
the evidence for the presence of several repeats and hinges of the rod
domain between the amino- and carboxyl-terminal ends, which is
essential for a complete rescue of the dystrophic phenotype of the
mdx mouse (14). The above studies lead to the view that the
rod domain may target the dystrophin molecule onto the sarcolemma by a
lateral association not only with F-actin but also with membrane
phospholipids. Interactions with membrane lipids have already been
reported for spectrin (15, 16) and have been shown to be involved in
the shape regulation of the red cell membrane (17) and more recently in
its membrane stability (18). Because spectrin and dystrophin belong to
the same protein family, these results suggest a similar function for
the two proteins.
However, the nature of the association of the rod domain with membrane
lipids is still unclear. Tryptophan (Trp) residues are often involved
in the membrane lipid association of interfacial proteins (19-21).
Because several tryptophan residues are present in the rod domain
repeats, we investigated these tryptophan properties and accessibility
in two recombinant proteins of the rod domain, both in aqueous solution
and in the presence of membrane lipids.
Calvert et al. (22) have made several interesting constructs
of the second repeat of the human dystrophin rod domain involving a
constant amino-terminal end but with variable carboxyl-terminal ends.
In the present study, we chose to investigate two of these fragments,
namely, the 114- and 125-residue-long proteins. Four Trp residues are
present in the 114 protein, whereas there is one extra Trp residue in
the 125 fragment (10). Although the 114 fragment does not fully fold,
proteins longer than the 117 residues such as the 125 fragment are able
to adopt a high percentage of We first used 1H NMR spectroscopy, a technique that can
give indications about the shielding of Trp residues in proteins. In addition, magnetization transfer between water and amide protons of Trp
residues (24) may provide information about their respective accessibility to water. In addition, intrinsic fluorescence
spectroscopy of tryptophan (Trp) is a valuable tool for studying the
conformation of proteins by means of time-resolved fluorescence and
quenching experiments (25-27). This can provide information about the
environment of the indole ring of Trp, including its charge and
polarity. Comparative Trp fluorescence spectroscopy of the two
fragments of dystrophin rod domain proteins having different folding
states can be used to determine the status of the Trp residues in these proteins and assess the possibility of their involvement in an interaction with membrane phospholipids.
Protein Preparation--
Proteins with lengths of 114 (F114) and
125 (F125)1 residues of the
second repeat of human dystrophin rod domain were prepared by
expression in Escherichia coli (strain BL21(DE3)) of
plasmids kindly provided by W. B. Gratzer (22). The amino terminus
is common to the two proteins and taken at the 439th residue of human dystrophin (NCBI Protein Data base NP_003997), and the C terminus ends
were at the 553rd and 564th residues for F114 and F125, respectively. Trp residues are situated at positions 25, 102, 108, and 112 for F114,
whereas a fifth is situated at position 123 in F125. Expressed proteins
were recovered as inclusion bodies from the E. coli
expression strain and purified as described previously (28). The
proteins were recovered by dispersion in 6 M guanidinium
chloride, 10 mM dithiothreitol and then purified by gel
chromatography in the same solvent on Sephacryl-S100. Fractions were
screened by UV absorption at 280 nm and SDS-gel electrophoresis. The
purified protein was kept in guanidinium chloride and dialyzed when
required against the desired buffer, typically 150 mM NaCl,
50 mM sodium phosphate, pH 7.6 (buffer A). Protein
concentration was determined spectrophotometrically at 280 nm using
molar extinction coefficients of 23,350 and 28,570 cm Preparation of Phospholipidic Vesicles--
Multilamellar
vesicles were first prepared. Mixtures containing a 2:1 molar ratio of
the lipids DOPC2 and DOPS or
DOPE in chloroform were dried overnight under vacuum and suspended in
solution A containing NaCl 150 mM, EDTA 0.1 mM buffered with Tris-HCl (100 mM), pH 7.6. Small unilamellar
vesicles (SUVs) were prepared extemporaneously from
multilamellar vesicles diluted at 25 mg/ml and subjected to sonication
at room temperature with the micro-tip of a sonicator (U200S, UKA
Labortechnic) for 5 min with half-duty cycles. They were then
centrifuged to eliminate titanium impurities. For fluorescence
quenching experiments with membrane-bound quenchers, SUVs containing
10% brominated phosphatidylcholine (BrPSPC, dibromine
palmitoylstearoylphosphatidylcholine), i.e. with the
composition DOPC/DOPS/BrPSPC (1.9:1:0.1, M/M),
were prepared. BrPSPC is commercially available (Avanti Polar,
Birmingham, AL) and brominated at either 6 and 7, or 11 and 12, of the
stearoyl acyl chains. For these SUVs, sonication was reduced to 1 min
to avoid degradation of the spin label (29). SUVs with 10%
non-brominated PSPC were used as control.
Circular Dichroism Measurements--
Circular dichroism was
measured (Jobin-Yvon CD6) at 281 K, with a path length of 0.02 cm and
at a concentration of 50 µM protein. The percentage of
molar ellipticity was calculated using a 100% 1H NMR Spectroscopy--
1H NMR spectra
were acquired at 500 MHz on a Bruker DMX spectrometer (11.5 teslas)
equipped with a 5-mm probe (Bruker Spectrospin, Wissembourg, France).
Protein concentration was 200-500 µM in the presence of
3% D2O. Spectral width was 12 ppm, and the number of scans
was 512-1024, using a repetition delay of 2 s with a 90° pulse
angle. Proton decoupling sequences were used with a solvent
presaturation or a WATERGATE sequence. Fourier transform was applied
after a line broadening of 0.2 Hz. Chemical shifts were referenced
against DSS by means of residual water resonance. Progressive
magnetization transfer between water and NH protons was measured at 313 K with variable solvent presaturation delays (0.25, 0.5, 1, and 2s).
The degree of magnetization transfer was estimated from the peak areas measurements.
Steady-state Fluorescence Measurements--
Tryptophan
fluorescence emission spectra were recorded between 320 and 450 nm
using an excitation wavelength of 295 nm (bandwidth, 8.4 nm) on a SPEX
112 spectrofluorometer (Jobin-Yvon, Longjumeau, France), using a 10- × 10-mm quartz cuvette at 293 K. Blanks were always subtracted in the
same experimental conditions (buffer, SUVs). Fluorescence intensities
were obtained by integrating the spectra in the range 320-450 nm.
Quantum yields were calculated by comparing the fluorescence
intensities of the proteins with a solution of DL-Trp in
buffer A, corrected to the same absorption at 295 nm (i.e.
the excitation wavelength). A value of 0.13 was used for the quantum
yield of free Trp (26).
Fluorescence quenching data were obtained with I
In the case of dynamic quenching, we obtained a linear array of points
on the plots. The Stern-Volmer constant (Ksv) is
derived from the slope of these plots. However, in some experiments,
the plots deviated upward from linearity, and this could be explained by the existence of both static and dynamic quenching. Because of this,
we used a modified Stern-Volmer equation (26),
Titration of the Trp fluorescence emission of F125 with increasing
concentrations of SUVs was effected as follows: small-volume aliquots
of stock SUVs (31 mM total lipids) were successively added
to F125 1 µM in a quartz cuvette. After each addition, 15 min of incubation was allowed under gently stirring before spectrum acquisition. Fluorescence intensity was determined by integrating spectra from 320 to 450 nm. Control spectra from SUVs alone were performed under the same conditions and subtracted from the respective protein spectra. When fluorescence spectra were acquired with SUVs
containing brominated phosphatidylcholine, the depth of insertion was
calculated using the "parallax" analysis (29, 30) with the
following equation,
Time-resolved Fluorescence Measurements--
Fluorescence
intensity decays were obtained by the time-correlated single photon
counting technique from the Ivv(t) and
Ivh(t) components recorded on the experimental
setup installed on the SB1 window of the synchrotron radiation source
Super-ACO (Anneau de Collision d'Orsay), which has been described
elsewhere (33). The excitation wavelength was selected by a double
monochromator (Jobin-Yvon UV-DH10, bandwidth 4 nm). An MCP-PMT
Hamamatsu instrument (model R3809U-02) was used for the fluorescence
measurements. Time resolution was around 20 ps, and the data were
stored in 2048 channels. Automatic sampling cycles included 30-s
accumulation time for the instrumental response function and 90-s
acquisition time for each polarized component. The sampling was carried
out such that a total number of 2-4 × 106 counts was
attained in the fluorescence intensity decay. For measurements in the
presence of SUVs, a cut-off filter (300 AELP filter, Omega Optical,
Brattleboro, VT) was used to minimize contamination of the fluorescence
decays by scattered excitation light. Analyses of fluorescence
intensity decay as exponential sums were performed by the maximum
entropy method (33).
Circular Dichroism Spectra--
The circular dichroic spectra of
the purified proteins F114 and F125 measured at 281 K exhibit molar
residue ellipticities at the extremum (222 nm) of 1H NMR Spectra--
The 1H NMR spectra
obtained from the two proteins are shown in Fig.
1. The F114 spectrum corresponds to an
only partly folded protein as indicated by the unresolved signals,
particularly for the NH resonances. In contrast, the F125 spectrum
displays a high chemical shift dispersion that is characteristic of
folded proteins. A good marker of the tertiary structure is the
presence of the two high-field shifted resonances at
The 1H NMR spectra of F125 show only slight changes between
278 and 313 K (data not shown). This observation is in good agreement with the reported temperature of half denaturation of 347 K (22). Nevertheless, the signals labeled 1-5 in Fig. 1 become
modified at 313 K compared with 298 K (Fig.
2). It is known that, under conditions of
relatively fast exchange, hydrogen exchange rates can be measured by
NMR spectroscopy to access information about the solvent accessibility
and the participation of exchangeable hydrogen within hydrogen bonds
(24). When using a presaturation sequence, we observe a signal decrease
for several of these protons by comparison with the spectra acquired
with the WATERGATE sequence (Fig. 1). This decrease of signal for some
hydrogen from the NH region indicates that, at 313 K, there is an
efficient magnetization transfer between water and NH protons during
the presaturation delay. We therefore investigated the magnetization
transfer occurring in F125 between water protons and NH protons at 313 K, using variable delays of presaturation. The results are shown in
Fig. 2. It appears that there is no magnetization transfer on
peaks 1 (10.75 ppm) and 2 (10.18 ppm). By
contrast, peaks 3-5 (10.12 ppm) are strongly reduced by the
presaturation of water. Because peaks 1, 2, and 3-5 are derived from the five Trp residues of F125, it
appears that at least two Trp residues out of five show no significant exchange with water protons at 313 K and pH 7.5. We did not attempt to
study the temperature dependence of the exchange with F114, because
this protein is known to have a half-denaturation temperature lower
than 300 K (22).
Spectra and Decay Times of Trp Fluorescence Emission from F114 and
F125--
Fluorescence emission spectra acquired with an excitation
wavelength of 295 nm were centered at 352 nm for F125 and at 356 nm for
F114, compared with the maximum at 362 nm for a DL-Trp solution obtained in buffer A (Table I).
The measured quantum yield was 0.07 and 0.05 for F114 and F125,
respectively, taking a value of 0.13 for Trp in solution (34).
The fluorescence decay of each protein was also observed (see Fig.
3A, for an example). The
curves are different for the two proteins and could not be approximated
by a single-exponential function. Using the maximum entropy method, the
decays are well fitted by three lifetime populations for F125 and F114
(Fig. 3B, upper part). Overall, average lifetimes
were calculated as 1.4 and 2.2 ns for F125 and F114, respectively
(Table I). These results are in good agreement with the quantum yield
values and indicate that the folded state of F125 partly "buries"
some Trp residues in a more hydrophobic environment than in F114.
The anisotropy decays of each protein exhibited two different
correlation time patterns (Fig. 3, A and B,
lower part). For F125, we obtained only one correlation time
at 6.8 ns (Fig. 3, lower part). This value is characteristic
of Brownian mobility for a compact globular protein of the size of F125
(14.9 kDa). By contrast, we obtained three rotational correlation time
peaks for F114 at 0.18, 1.4, and 5.2 ns (Fig. 3, lower
part); the two shorter peaks are characteristic of the high
mobility of the indole ring in the protein, whereas the longest peak is
characteristic of the Brownian mobility of a protein of about 13 kDa.
These results show that F125 is a structured protein, whereas F114
displays some highly mobile elements and is not as compact as F125.
As a whole, these results show that F125 is a highly structured protein
compared with F114. Because of this, it could be considered as a
control representing a poorly folded protein for the subsequent experiments. It was not possible to use denaturated F125 as the unfolded control, because when denaturated, F125 was not more water-soluble.
Steady-state and Time-resolved Fluorescence Quenching of Trp
Emission of the Two Proteins in Aqueous Solution--
Trp fluorescence
quenching is a valuable method for comparing the accessibility of Trp
residues to the solvent for the two studied proteins. We used
Cs+, I
Quenching by I
For acrylamide quenching, we found a static component for both of the
proteins. The static constant, V, and the Stern-Volmer constant, K, were both significantly higher for F114 (values
of 1.4 ± 0.06 and 6.5 ± 0.1 for V and
K, respectively) than for F125 quenching (values of 0.9 ± 0.2 and 5.1 ± 0.4 for V and K,
respectively) (results not shown).
We found no evidence of static quenching by TCE for F125,
in contrast with F114 that yielded a static constant of about 3 M
Due to the presence of static quenching observed with acrylamide and
TCE, we obtained curves of
By contrast, TCE quenching curves were different for the two proteins,
F114 displaying a higher Ksv value than for F125
(Table III). These were two to three
times lower than the values measured by steady-state fluorescence,
indicating the existence of a much more efficient static quenching for
F114 than for F125. In addition, the accessibility and exposure of Trp
residues to TCE are larger in the former protein than in the latter.
High values of TCE quenching have been reported for proteins known to
have hydrophobic pockets able to bind small lipidic molecules such as
the bovine (Ksv = 230 M Effects of Membrane Phospholipids on Trp Fluorescence Emission of
Both Proteins--
In a previous study, DeWolf et al.
(10) showed that a fragment of 119 residues (F119) modified the
physical properties of a model membrane containing anionic
phospholipids, indicating that there was an interaction between the
fragment and the phospholipids. This effect was much weaker for the
fragment of 114 residues. To obtain further insight into this
interaction, we incubated F125 (not used by DeWolf) and F114 with SUVs
made up from the DOPC/DOPS or DOPC/DOPE 2:1 mixtures. These membrane
models were chosen because the interference of fluorescence emission
was minimal between Trp and these small vesicles. The incubation
conditions were similar to those used by DeWolf, i.e. the
ratio between proteins and SUV was 1 µM protein for 1.5 mM lipids, and the incubation was performed for 3 h at
pH 7.5 in the presence of 0.15 M NaCl. Fig.
4A shows that the fluorescence
intensity for F125, measured in the spectrum range between 320 and 450 nm, increases exponentially as a function of time in the presence of
DOPC/DOPS SUVs. A maximum F/F0
value of about 1.9 was observed after 3 h. At the same
time, the
We further examined the influence of an increased concentration of
total lipids on the Trp fluorescence emission of F125. We acquired
successive spectra of 1 µM F125 in the presence of increasing concentrations of DOPC/DOPS. Fig.
5 shows that the effect of SUVs on the
Trp fluorescence intensity is subject to saturation, with a maximum
fluorescence enhancement of about 1.9, as observed in Fig. 4.
The fluorescence decay of both proteins in the presence of DOPC/DOPS
SUVs can be described by the sum of three exponential components (Fig.
6), whereas the average lifetimes
All these modifications of F125 Trp fluorescence in the presence of
anionic SUVs are indicative of a more hydrophobic environment of the
Trp residues. This suggests either the existence of contacts in the
vicinity of the indole rings between the protein F125 and the membrane
lipids (26), or a conformational change burying most of the Trp
residues inside the protein. Either of these effects is likely to be
very moderate for F114. Thus, the effects of the presence of anionic
SUVs appear to be much more specific in the case of the more structured protein.
Effects of SUVs on Trp Fluorescence Quenching of F125--
The
existence of contacts between Trp residues and membrane lipids implies
that the accessibility of Trp residues in F125 in the presence of
anionic SUVs must be decreased compared with free F125. The
accessibility of Trp residues in F125 in the presence of DOPC/DOPS SUVs
was therefore explored here by fluorescence quenching. Steady-state
fluorescence quenching was carried out using Cs+,
I
The bimolecular quenching constant of TCE for F125 in the presence of
DOPC/DOPS SUVs is five times higher than in water (Table III). This
enhanced quenching by TCE is also observed by steady-state fluorescence, where the Ksv values are 13 and 64 M
We also checked the TCE partitioning within hydrophobic volumes such as
membrane systems using a model system of dodecyl-maltoside micelles
that comprise a long-tailed tryptophan derivative, the tryptophanyl-octyl ester. The fluorescent moiety of this ester is known
to be located at the polar-hydrophobic interface (37). The
kq value associated with tryptophanyl-octyl
ester quenched by TCE was measured at 19 M
In addition, by comparing this strong effect of TCE on F125 in the
presence of DOPC/DOPS SUVs, we can see that the
kq value obtained by time-resolved fluorescence
of F114 was not modified by the presence of these SUVs (Table III).
This demonstrates that Trp fluorescence of F114 is unaffected by SUVs
and, thus, that the strong effect of the presence of DOPC/DOPS SUVs on
F125 is specific of a structured protein. The overall results from TCE quenching show that the Trp residues of F125 in the presence of DOPC/DOPS SUVs are in a more hydrophobic environment than in F125 in
aqueous solution and are probably in close proximity to the membrane lipids.
Trp Fluorescence Quenching of F125 with Membrane-bound
Quenchers--
Additional support for the close proximity of F125 to
the membrane lipids comes from two other data on quenching of
fluorescence with lipids brominated or spin-labeled at different
positions on the acyl chain, i.e. 5-doxyl and 16-doxyl
stearic acid and 6,7-diBrPC- and 11,12-diBrPC-containing SUVs. Because
these lipids are membrane-bound quenchers, comparison of their
respective quenching ability yields information about the relative
location of the tryptophan residues compared with the surface of the bilayer.
With 5-doxyl and 16-doxyl stearic acid (Fig.
7A), calculation of the
apparent Stern-Volmer constant revealed that 5-DSA, with an apparent
Stern-Volmer constant of 7 ± 1 mM
A similar conclusion came from experiments where F125 was incubated
with SUVs comprising 10% of brominated PC (Fig. 7B). It appears that the presence of brominated phospholipids in the vesicles induced significant quenching of the Trp fluorescence in F125. The
magnitude of the quenching is higher when the bromine atoms are located
in positions 6 and 7 of the acyl chain with 42% of quenching
efficiency, than when present at positions 11 and 12 with 16% of
quenching efficiency, and is consistent with a location of the protein
close to the shallow quencher. We attempted to calculate the depth of
penetration of Trp residues by the "parallax" method, according to
the expression given under "Materials and Methods," being aware
that the computed value of Zcf is an average over the five Trp residues of the F125. A value of 18.5 Å is obtained for the distance between the Trp and the center of the bilayer. The
main conclusion is that the protein Trp residues are, on the average,
located closer to the membrane/water interface than the 6 to 7 bromine
atoms. We thus conclude that the protein is lying along the membrane
surface with several Trp residues very close to the polar head groups
of the phospholipids.
The function of the rod domain of dystrophin, which is made up of
24 spectrin-like repeats, remains unresolved. Two interesting observations indicate that some central basic repeats bind to F-actin
(6) and that the second repeat may interact with artificial membrane
containing phosphatidylserine (10). This latter observation provides
some clues about the targeting of the dystrophin onto the sarcolemma.
Thus, it remains important to determine the nature of the interaction
between membrane phospholipids and the dystrophin rod domain. We need
to determine whether the Trp residues (five in the second repeat) are
involved in the interaction. The present study supports the view that
Trp residues become located at the water-membrane interface in the
complex protein-membrane and that this process concerns only the folded
protein F125.
Various lines of experimental evidence demonstrate that the longer
protein (F125) is more folded than the shorter (F114). The high
chemical shift dispersion in the 1H NMR one-dimensional
spectrum and the higher fluorescence maximum emission intensity, as
well as the shorter excited-state lifetime and the blue shift of the
Trp fluorescence emission of F125 compared with F114, all indicate that
the Trp residues of F125 are more shielded from the surface than in the
case of F114. This view is accepted as the general rule for folded
proteins (26). The anisotropy decay confirms that F125 is more compact
and structured than F114.
Overall, the fluorescence quenching data reported here
indicate that both of the proteins in aqueous solution are
characterized by structural fluctuations. In fact, all the Trp residues
in F125 and F114 appear to be accessible to fluorescence quenchers. The lower quenching efficiency of the Cs+ ions compared with
the I Finally, the non-polar TCE quenches the fluorescence of Trp residues in
close proximity to hydrophobic sites at the surface of the proteins
(35, 36). Thus, it is a more effective quencher for F114 than for F125,
indicating that the surface of the folded protein F125 is relatively
less hydrophobic than F114.
Several strong changes occur when F125 is in the presence
of model membranes made up partly of anionic phosphatidylserine. On the
other hand, very weak changes are observed for F114 with SUVs or for
F125 with zwitterionic SUVs. In the presence of anionic SUVs, the
maximum intensity of the fluorescence emission spectrum is increased
and shifted toward shorter wavelengths compared with the free F125
protein, indicating that the Trp residues are located in a more
hydrophobic environment in the complex F125·SUVs (43-47). In fact,
this modified localization of the Trp residues could be due either to
the close proximity of indole rings to the membrane phospholipids or to
a protein conformational change that is able to bury the Trp residues
from the surface. However, the latter effect would be
accompanied by a shortening of the fluorescence excited-state lifetime
of the Trp emission as is often observed in structured proteins (26).
This is not the case here, because the excited-state lifetime is
largely increased in the complex F125·SUVs compared with the free
F125, in agreement with the 2-fold increase of fluorescence intensity.
Such observations are the general rule when Trp residues are close to
the surface of the lipid membrane and not fully exposed to the aqueous
solvent. This indicates clearly that the more hydrophobic environment
encountered by Trp in the complex F125·SUVs is produced by the close
proximity of the Trp to the membrane surface (42). It is noteworthy
that these large modifications are restricted to the highly folded repeat F125, because the poorly structured protein F114 detects only a
very weak modification when in the presence of SUVs.
The large decrease of Trp fluorescence quenching by ions and acrylamide
observed in the complex F125·SUVs compared with the free F125 shows
that the Trp residues are shielded from the solvent and confirms the
localization of Trp residues at the membrane surface. By contrast, we
see a large increase in the TCE quenching of Trp residues in the
F125·SUV complex. Because TCE have the property of partitioning into
hydrophobic pockets at the surface of proteins, where it dynamically
quenches the fluorescence emission, the quenching is enhanced in a
hydrophobic environment (35). The 6-fold increase in the bimolecular
TCE quenching constant of Trp residues in the F125·SUV complex thus
shows that the Trp residues are in a highly hydrophobic environment.
Overall, our observations clearly suggest that the formation of a
complex between F125 and SUVs leads to a shielding of Trp residues from
the aqueous solvent by a mechanism that places them in close contact
with the membrane phospholipids.
Trp residues in membrane or peripheral proteins tend to cluster near to
or at the lipid-water interface of the membrane (20, 48). In membrane
proteins, Trp residues are mostly located in extramembranal segments
lying parallel to the surface of the membrane. Aromatic residues such
as Trp, Tyr, and Phe are known to be particularly suitable for
interfacial positioning, because they can effectively bridge the gap
between the two contrasting environments existing at the membrane-water
interface (19, 49). In the repeat of the dystrophin rod domain, it is
likely that several Trp residues are also accommodated at the
lipid-water interface. This is strongly supported by the data obtained
with membrane-bound quenchers. The fact that these compounds are
effective quenchers showed that the Trp residues are not far from these
quenchers. In addition, the quenching efficiency was more pronounced
with the shallow quenchers, 5-DSA and 6,7-diBrPC, than with the deeper
quenchers, 16-DSA and 11,12-diBrPC, indicating unequivocally
that the Trp residues are, on the average, located at the surface of
the bilayer. Four out of the five Trp residues of F125 are situated in
the presumed third helix of the repeat, so we infer that the repeat lies along the surface of the membrane bilayer. However, Trp
fluorescence modifications are not observed for SUVs prepared with
zwitterionic phospholipids alone, i.e. when no net charges
are present at the membrane surface. This suggests that hydrophobic
interactions between Trp residues and the membrane lipids are not the
driving force behind the formation of the F125·SUV complex. It is
likely that electrostatic attractions play a role in the initiation of complex formation, which could then be stabilized by both hydrophobic and electrostatic forces as in the case of many peripheral membrane proteins (48). Dystrophin may thus be classified in the important family of lipid-binding cytoskeletal proteins, which includes vinculin,
spectrin, and talin, proteins also known to bind other cytoskeletal
proteins (actin, Finally, our study stresses the potential role of the rod domain of
dystrophin in targeting onto the sarcolemma, even when the
carboxyl-terminal end is absent as observed in patients with genetic
variants lacking this end (11-13). Furthermore, the presence of
several repeats and hinges is essential for a complete rescue of the
mdx phenotype (14, 51). The dystrophin molecule appears to
be involved in an important array situated at the sarcolemma, comprising not only cytoskeletal proteins (5, 6, 52) and membrane
proteins (53-56) but also membrane phospholipids.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helical content (22, 23).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1
M
1 for F114 and F125, respectively (23).
-helix coefficient of
36,000 deg·cm2·dmol
1 at 222 nm
(23).
(KI),
Cs+ (CsCl), 2,2,2-trichloroethanol (TCE) and acrylamide as
follows: small-volume aliquots of stock solutions at 5 M of
KI, CsCl, and acrylamide prepared in the protein buffer A and at 1.3 M for TCE in ethylene glycol were successively added to the
samples. After each addition, the solution was gently stirred and the
decrease of fluorescence intensity was measured at the initial
max. A spectrum was recorded at the end of each series
to check that the quencher had no effect on the
max of
the protein. As controls, KCl and ethylene glycol were shown to have no
effect on the fluorescence of the proteins. Appropriate corrections for
dilution were made for these experiments. The same procedure was used
for quenching experiments with 5- and 16-doxyl stearoyl acids dissolved
as a 20 mM stock solution in Me2SO
except that a 5-min incubation delay was allowed before data
acquisition after each 1-µl addition. Effective Stern-Volmer
constants (Ksv) were obtained from the fluorescence data according to the Stern-Volmer equation for dynamic quenching (in which collisional quenching is mainly governed by diffusion processes) (25, 26),
where F0 and F are the
fluorescence intensities without and with the presence of the quencher
Q, respectively, kq is the bimolecular collisional constant, and
(Eq. 1)
0 is the life time
constant in the absence of quencher.
where K is the constant for dynamic quenching and V
the constant for static quenching. Concerning quenching experiments
with doxyl stearic acids, which are not soluble quenchers,
"apparent" Stern-Volmer constants were calculated with Equation 1.
(Eq. 2)
where Zcf is the transverse distance from
the plane of the bilayer center to the plane containing the Trp
residues, F1 and F2, are fluorescence intensities
in presence of the shallow quencher and deeper quencher, respectively,
Lc1 is the transverse distance from the bilayer
center to the shallow quencher, L12 is the
transverse distance between the depths of the two quenchers, and
C is the two-dimensional quencher concentration in the plane
of the membrane in mole fraction of quencher lipid in total lipid per
unit area. The values for the constants were Lc1 = 10.8 Å, L12 = 4.5 Å for the distance between
6,7- and 11,12-diBrPC according to x-ray diffraction (31);
C = (0.1)/70 Å2, the average surface area
of quencher (32). All the experimental data were fitted to the cited
equations using non-linear regression analysis with Sigma-Plot software
(Jandel Scientific).
(Eq. 3)
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
18,000 and
30,000
deg·cm2·dmol
1, respectively. This
correspond to
-helicities of 50 and 83% for F114 and F125, respectively.
0.03 and
0.19
ppm, as well as the sharp proton signals between 11.5 and 9.5 ppm.
These resonances, labeled 1-5, are assigned to the NH
indole protons of the five Trp residues of F125.
View larger version (22K):
[in a new window]
Fig. 1.
1H NMR spectra of F114 and F125
at pH 7.5 recorded at 500 MHz and 298 K with the WATERGATE
sequence. The numbers 1-5 refer to Trp residues with
the Trp-1 arising at 10.6 ppm, Trp-2 at 10.08 ppm, Trp-3 and -4 at 10.0 ppm, and Trp-5 at 9.95 ppm.
View larger version (20K):
[in a new window]
Fig. 2.
1H NMR spectra of F125 at pH 7.5, 500 MHz, and 313 K. Four spectra were acquired with a
pre-saturation sequence for solvent suppression with respective delays
of 0.25, 0.5, 1, and 2 s as indicated. The spectrum in the
upper part was acquired using a WATERGATE sequence for
solvent suppression.
Fluorescence emission spectroscopy characteristics of a
DL-Trp solution in buffer A compared to the two proteins
with and without SUVs
View larger version (24K):
[in a new window]
Fig. 3.
A, experimental fluorescence intensity
(o) and anisotropy decays ( ) of Trp residues in
F125. Protein concentration was 1 µM. The
excitation and emission wavelengths were 295 and 350 nm, respectively.
The instrumental response function is indicated as a dotted
line. B, excited-state lifetime distribution profiles
(upper part) and rotational correlation time distribution
profiles (lower part) of Trp residues in F125 and F114. The
excitation wavelength is 295 nm, and the emission wavelengths were 352 nm for F125 and 356 nm for F114. Protein concentration was 1 µM.
, acrylamide, and
2,2,2-trichloroethanol (TCE) as quenchers of Trp residue fluorescence.
and Cs+ yield linear plots of
F0/F versus [Q], whereas
max was unaffected by the addition of these quenchers. The dynamic apparent quenching constants Ksv are
derived from the slopes of each of the Stern-Volmer plots (Table
II). The Stern-Volmer quenching constants
for Cs+ and I
are significantly higher for
F114 than for F125. However, the bimolecular quenching constant,
kq, calculated using the average
0 obtained by time-resolved fluorescence, is
slightly lower for F114 than for F125 (Table II). Generally speaking,
these results show rather low bimolecular quenching constants compared
with the values of 6-7 M
1 ns
1
for free indole derivative.
Trp fluorescence emission quenching of F114 and F125 in aqueous
solution, and F125 in the presence of SUVs, using cesium and iodide
ions and acrylamide
1. The apparent dynamic constant is very
high at 20 ± 4 M
1 for F114 compared
with 9 ± 1 M
1 for F125. The value of
max was unaffected by the addition of the quenchers
(results not shown).
0/
and the dynamic
bimolecular quenching constant, kq, from
time-resolved fluorescence measurements using these quenchers. For
acrylamide quenching, the curves are similar for F114 and F125 leading
to KSV values around 3.5 M
1 (Table II). These quenching constants are
twice as low as those measured by steady-state fluorescence, indicating
that the static quenching component cannot be accurately subtracted
from the steady-state fluorescence data. The bimolecular quenching
constant kq is slightly higher for F125 compared
with F114 (Table II). Overall, these results indicate a rather similar
accessibility of Trp residues to acrylamide in both proteins.
1) or human serum albumin
(Ksv = 20 M
1) (35).
This is also the case for the whole spectrin molecule with a
Ksv value of about 20 M
1 (36). By contrast, the
Ksv for F125 is similar to that obtained for
acrylamide quenching, leading to the idea that Trp residues are not
more accessible to TCE than to acrylamide in the folded protein F125.
Such a conclusion can be drawn for most of the proteins.
Time-resolved Trp fluorescence quenching of the two proteins in aqueous
solution and in the presence of SUVs, using TCE
max shifted by 10 nm, from 352 to 342 nm (Fig.
4B). When DOPC/DOPE was used instead of the DOPC/DOPS
mixture, there was only a very weak increase of fluorescence intensity
and no
max shift (Fig. 4, A and
B), indicating that the F125 fluorescence modifications upon
addition of DOPC/DOPS SUVs was due to the presence of DOPS. We observed
a weak effect of DOPC/DOPS SUVs on F114 characterized by a rapid
increase to 1.2 of the initial fluorescence and a further progressive
decrease to 1.10 after three hours (Fig. 4A). The
max
shifts by 6 nm from 356 nm to 350 nm (Fig. 4B).
View larger version (18K):
[in a new window]
Fig. 4.
A, time evolution of fluorescence
emission of 1 µM of F125 and F114 in the presence of 1.5 mM DOPC/DOPS 2:1 (M:M) SUVs
(filled triangles and white circles,
respectively) and of 1 µM F125 in the presence of 1.5 mM DOPC/DOPE 2:1 (M:M) SUVs
(white triangles). The excitation wavelength was 295 nm,
with fluorescence intensity measured as the area of the spectra from
320 to 450 nm. The SUVs control spectra are subtracted from each data
obtained with the proteins. B, fluorescence spectra of the
preparations presented in A. Conditions were similar to
A.
View larger version (10K):
[in a new window]
Fig. 5.
Variation of the Trp fluorescence emission of
1 µM F125 as a function of the phospholipid
concentration (DOPC/DOPS 2:1 M/M). The
line represents the fit to a hyperbolic function.
0 in the presence of SUVs are calculated as 2.6 and 2.8 ns for F125 and F114 (Table I), respectively. Thus, upon interaction
with anionic SUVs, the lifetime for F125 increases by 85% as against
27% for F114. This is in agreement with the increases in the
fluorescence intensities observed under these conditions.
View larger version (20K):
[in a new window]
Fig. 6.
Excited-state lifetime of Trp residues in
F125 (upper part) and F114 (lower
part) after 3-h incubation with 1.5 mM SUVs
(DOPC/DOPS 2:1, M/M) (solid
lines). For comparison, the profiles obtained without
SUVs in Fig. 3 are shown (dashed lines). The excitation
wavelength was 295 nm, whereas the emission wavelengths were 342 and
350 nm for F125 and F114, respectively. Protein concentration was 1 µM.
, and acrylamide. The Ksv values
for Cs+ quenching decrease by 40% for F125 incubated with
DOPC/DOPS SUVs compared with the protein alone, yielding a bimolecular
quenching constant more than five times lower than in the absence of
membrane phospholipids (Table II). The same result holds for
I
and acrylamide quenching, with bimolecular quenching
constants decreasing about 5-fold and more than 3-fold, respectively,
in the presence of DOPC/DOPS SUVs as compared with aqueous solution (Table II). Thus, it appears that the presence of SUVs strongly decreases the exposure of the Trp to these quenchers, arguing for a
close proximity of the Trp to the membrane surface that is generally
able to shield Trp residues from the solvent.
1 for F125 in aqueous solution and in the
presence of DOPC/DOPS SUVs, respectively (data not shown). A large
static component is revealed by the discrepancies between the
Ksv measured by steady-state and time-resolved
fluorescence. The explanation for this enhanced quenching by TCE is as
follows: TCE partitions into the lipid bilayer, where it senses the
hydrophobic environment and efficiently quenches the Trp residues that
occur in this hydrophobic environment. These results clearly indicate
that the environment of the Trp residues is highly hydrophobic when
F125 is in the presence of DOPC/DOPS SUVs.
1
ns
1. This is six times higher than the value for the
water-soluble derivative N-acetyl-tryptophanamide, which
yielded a value of 3 M
1 ns
1
(results not shown). These results confirm those obtained with F125 in
the presence of DOPC/DOPS SUVs.
1, was
2-fold more effective in quenching fluorescence than 16-DSA, with a
constant value of 3.3 ± 0.2 mM
1. As a
control, 5-DSA quenched weakly the fluorescence of Trp of F125 in
presence of SUVs composed of zwitterionic SUVs, with an apparent
Stern-Volmer constant of 2.3 mM
1. This leads
us to conclude that Trp residues of F125 in the presence of anionic
SUVs are very close to the surface of the bilayer.
View larger version (20K):
[in a new window]
Fig. 7.
A, Stern-Volmer plots of quenching of
tryptophan fluorescence in F125 1 µM in the presence of
1.5 mM SUVs by doxyl stearic acids. Increasing
concentrations of 5-doxyl and 16-doxyl stearic acids (white
and black circles, respectively) were added to F125 in the
presence of anionic DOPC/DOPS SUVs. As a control, 5-doxyl stearic acid
was added to F125 in the presence of zwitterionic DOPC/DOPE SUVs
(white triangles). Excitation wavelength was 295 nm, and
emission wavelength was 342 nm. A plot of
F0/F versus doxyl stearic
acids yields a line where the slope is the apparent
Stern-Volmer constant. B, fluorescence spectra of 0.2 µM F125, in the presence of 1 mM DOPC/DOPS
2:1 SUVs where DOPC was substituted for 10% by
palmitoyl-stearoyl-phosphatidylcholine non-brominated (solid
line) or brominated in positions 11 and 12 (dotted
line) or 6 and 7 (dashed line), were recorded between
300 and 450 nm. Excitation wavelength was 295 nm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ions is consistent with a preferential cationic
environment for the Trp residues in both proteins. The acrylamide
bimolecular quenching constants, rather similar for F125 and F114, are
of the same order of magnitude as that found for a Trp residue in a
random coil polypeptide (38) as well as for Trp in entire spectrin (36)
and in a repeat of the c-Myb protein (39). These two last
proteins display a coiled-coil type structure (39, 40) as also assumed
for the repeats of the dystrophin rod domain (2, 22, 41). This
indicates that the Trp residues of coiled-coil proteins may be
relatively easily accessible to acrylamide. By contrast, the
accessibility of the studied proteins to acrylamide is one order of
magnitude higher than that reported for domain III of Annexin V, a
globular protein with a Trp residue deeply buried in the protein matrix
(42). In coiled-coil proteins, Trp are accessible either because of the
structure of the protein, which could be the case for the compact
folded F125, or, alternatively, due to large internal motions that
could be the case for the non-structured F114.
-actinin, and integrins) to form complex structural
arrays (50).
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ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge W. B. Gratzer for providing the plasmids and J. P. Douliez for acquisition of circular dichroism spectra. We thank F. Toma for stimulating discussions. M. S. N. Carpenter post-edited the English style.
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FOOTNOTES |
---|
* This work was supported in part by grants from the Association Française contre les Myopathies.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a fellowship from the Association Française
contre les Myopathies.
§ To whom correspondence should be addressed. Tel.: 33-2-23-23-46-27; Fax: 33-2-23-23-46-06; E-mail: elisabeth.lerumeur@univ-rennes1.fr.
Published, JBC Papers in Press, December 11, 2002, DOI 10.1074/jbc.M207321200
1 The F125 fragment was initially assumed to be F123, as proposed by DeWolf et al.. (4), but checking by mass spectroscopy and DNA sequence analysis showed that it is a F125 fragment, i.e. the carboxyl-terminal end is at the 564th residue of human dystrophin.
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ABBREVIATIONS |
---|
The abbreviations used are: DOPC, 1,2-dioleyl-sn-glycero-3-phosphocholine; DOPS, 1,2-dioleyl-sn-glycero-3-phospho-L-serine; DOPE, 1,2-dioleyl-sn-glycero-3-phospho-L-ethanolamine; 6, 7-diBrPC, 1-palmitoyl-2-stearoyl-(6,7)-dibromo-sn-glycero-3-phosphocholine; 11, 12-diBrPC, 1-palmitoyl-2-stearoyl-(11,12)-dibromo-sn-glycero-3-phosphocholine; PSPC, 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine; SUVs, small unilamellar vesicles; TCE, 2,2,2-trichloroethanol; 5-DSA, 5-doxylstearic acid; 16-DSA, 16-doxylstearic acid.
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REFERENCES |
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