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J Biol Chem, Vol. 273, Issue 51, 34214-34221, December 18, 1998
From the Department of Biology, University of California San Diego,
La Jolla, California 92093-0366
The assembly of target (t-SNARE) and
vesicle-associated SNAP receptor (v-SNARE) proteins is a critical step
for the docking of synaptic vesicles to the plasma membrane.
Syntaxin-1A, SNAP-25, and synaptobrevin-2 (also known as
vesicle-associated membrane protein, or VAMP-2) bind to each other with
high affinity, and their binding regions are predicted to form a
trimeric coiled-coil. Here, we have designed three peptides, which
correspond to sequences located in the syntaxin-1A H3 domain, the
C-terminal domain of SNAP-25, and a conserved central domain of
synaptobrevin-2, that exhibit a high propensity to form a minimal
trimeric coiled-coil. The peptides were synthesized by solid phase
methods, and their interactions were studied by CD spectroscopy. In
aqueous solution, the peptides were unstructured and showed no
interactions with each other. In contrast, upon the addition of
moderate amounts of trifluoroethanol (30%), the peptides adopted an
The assembly of the synaptic core complex is essential for
Ca2+-dependent neuroexocytosis. This early
event in the secretory cascade is then followed by the priming and
vesicle fusion steps (1-6). According to the
SNARE1 model, docking of
synaptic vesicles to the plasma membrane is a critical step that
involves the formation of a ternary complex by the v-SNARE
synaptobrevin (also known as vesicle-associated membrane protein, or
VAMP), and two t-SNAREs: SNAP-25 and syntaxin (7-9). Reconstitution of
the v-SNARE synaptobrevin into lipid vesicles and the two t-SNAREs,
SNAP-25 and syntaxin, into a distinct vesicle pool has provided
evidence that the formation of a ternary complex is sufficient to join
the independent vesicle pools and lead to fusion of the apposed bilayer
membranes (10).
Understanding the interactions between the proteins of the trimeric
complex in a simplified model may outline new ways to control its
assembly and dissociation or to modulate the conformational changes
that are presumably necessary for the progression from the docking step
to the subsequent phases in the secretory process. The structural
domains that appear to be implicated in the protein-protein interactions between SNAP-25, synaptobrevin, and syntaxin show a high
propensity for the formation of To investigate the postulated coiled-coil interactions between the
proteins that constitute the docking complex in a minimal model, we
have applied the principles involved in the formation of stable
coiled-coils (16) to design three peptides corresponding to predicted
coiled-coil-forming domains in SNAP-25, synaptobrevin-2, and
syntaxin-1A. We have used CD spectroscopy to determine the secondary
structure of these peptides and their interactions in binary and
tertiary mixtures. Our findings are consistent with the assembly of the
predicted ternary complex.
Reagents--
HPLC grade trifluoroacetic acid, trifluoroethanol
(TFE), ethanedithiol, thioanisol, phenol, and acetonitrile were
purchased from Aldrich. Methyl tert-butyl ether was from
Fisher. HPLC columns were from Vydac (Hesperia, CA).
L-Amino acids and protected derivatives used for peptide
synthesis were made by Calbiochem. Benzoic anhydride was obtained from
Sigma. All other reagents for peptide synthesis and resins were from
Applied Biosystems (Foster City, CA).
Peptide Synthesis and Purification--
Peptides SN (human brain
SNAP-25-(181-206)), SB (human brain synaptobrevin-2-(40-67)), ST
(human brain syntaxin-1A-(191-218)), and SNRD (scrambled
SN peptide sequence: ESDNDTRAIKITQAGSMKRMGLNAKE) were produced using
solid phase peptide synthesis. Synthesis started with a
p-hydroxymethyl phenoxy methyl polysterene resin and was carried out using the Fastmoc® Fmoc strategy on an Applied
Biosystems peptide synthesizer model 431A (Foster City, CA) according
to a single coupling plus capping protocol. Cleavage from the resin and
removal of all protecting groups was accomplished by using
trifluoroacetic acid cleavage as described (17). Crude peptides were
precipitated from the trifluoroacetic acid mixture in cold methyl
tert-butyl ether and centrifuged, the supernatant was
discarded, and the remaining methyl tert-butyl ether was
removed under high vacuum at 0 °C for 3 h. Samples of crude
peptide (10-20 mg) were dissolved in 0.1% trifluoroacetic acid,
applied to a semipreparatory column (Vydac, C-18), and eluted at a flow
rate of 3 ml/min with a linear gradient of 90% acetonitrile in 0.1%
trifluoroacetic acid. Eluted peaks were monitored by absorbance
measurements at 214 nm, pooled, and lyophilized. Peptide purity was
assessed by RP-HPLC in an analytical column (Vydac, C-18).
Secondary and Tertiary Structure Predictions--
Propensities
of peptides to adopt a coiled-coil structure were estimated using two
different programs: Coils and Paircoil. The Coils program uses the
Lupas algorithm (18, 19). Sequences were compared with an unweighted
MTIDK matrix (18) using 14- and 28-residue scanning windows. The second
program uses the Berger algorithm (20), which is more stringent. Both
methods are based on the relative frequency of occurrence of amino
acids at each position (a-g) of the coiled-coil heptad
repeat. Secondary structure predictions were performed using the SOPMA
method (21, 22) and the AGADIR program (23).
CD Measurements--
CD measurements were carried out on a
modified Cary 61 (24) or an AVIV model 202 spectropolarimeter. The
original Pockel cell and Cary linear polarizer in the Cary 61 were
replaced with a 50-kHz photoelastic modulator (Hinds International,
FS-5/PEM-80) and a MgF2 linear polarizer (AVIV Inc.). The
phase-detected output of the original end-on photomultiplier and
preamplifier were integrated using an Egg Princeton Applied Research
model 128A lock-in amplifier. System automation and multiple scan
averaging were accomplished with an IBM PC-compatible computer
interfaced directly to both the Cary 61 and the 128A amplifier.
Constant N2 flushing was employed. Spectra were measured at
195-250 nm using a 0.05-cm cell, a 1-nm bandwidth, a 0.3-ms time
constant, and a cell temperature of 25 °C. All recordings were
performed in 10 mM sodium phosphate buffer, pH 7.4, 100 mM NaCl, with or without TFE, unless otherwise indicated. Twenty scans were averaged for every spectrum. Base line subtraction, conversion of measured rotations to mean residue ellipticity [ Peptide Design
Basic Criteria for the Design of the Minimal Predicted
Coiled-coil-forming Peptides--
The sequences of the peptides
synthesized from selected regions from human SNAP-25 (peptide SN),
synaptobrevin-2 (peptide SB), and syntaxin-1A (peptide ST) are shown in
Fig. 1A. Sequence selection
was based on six criteria: 1) information about the minimal domains of
SNAP-25, synaptobrevin, and syntaxin involved in protein-protein
interactions in the core complex (1-5, 28); 2) botulinum neurotoxins
(BoNTs) cleavage sites and their effects on neurotransmitter release
(29-37); 3) sequence specificity of peptides inhibitors of
neurotransmitter release (38-44); 4) effects of point mutations on the
process of secretion and endocytosis (4, 11, 44-47); 5) predictions of
secondary structure formation of coiled-coil structures; and 6) a
minimum length for a stable parallel coil peptide of ~28 residues, or
4 heptad repeats (48, 49).
Design of the SN Peptide--
The region of SNAP-25 interacting
with synaptobrevin-2 has been localized between residue 41 and the
C-terminal residue (2, 4). The segment from residue 181 to the C
terminus is necessary for the SNAP-25-synaptobrevin interaction (3).
Peptides corresponding to the 20 and 26 C-terminal residues, the latter
analogous to the SNAP-25 segment released after cleavage by BoNT E,
inhibit neurotransmitter release with IC50 values of 10 and
0.25 µM, presumably by preventing the docking of synaptic
vesicles (40, 43).
The C-terminal region of SNAP-25 delimited by residues 169 and 206 displays a high propensity (99%) to form coiled-coil structures. Two
distinct domains are predicted: one from position 166 to 187 (62%),
and the second from position 189 to the C terminus (55%). Accordingly,
the 26-residue peptide corresponding to the C-terminal segment of
SNAP-25, hereafter designated as SN, was selected based on the fact
that it is nearly 4 heptads long, it exhibits high propensity to form
coiled-coils, and it is an efficient inhibitor of neurotransmitter
release (Fig. 1A, Peptide SN).
Design of the ST Peptide--
Similar considerations were used in
the design of a potentially coiled-coil-forming peptide from
syntaxin-1A. The region between positions 194 and 261 is necessary for
the interaction with synaptobrevin-2 and SNAP-25 (2, 3, 5). The segment
necessary for interaction with SNAP-25 has been located between
residues 199 and 267 (1) and further delimited to residues 199-220
(4). All of these studies confine the SNAP-25 binding region on
syntaxin to the N-terminal portion of the H3 domain (residues 191-266)
(2, 4), and a putative minimal SNAP-25 binding domain (residues 189-220) has been identified (11, 44). Coiled-coil predictions using
the sequence of human syntaxin revealed a region between positions 199 and 214 with high probability (78%) of coiled-coil formation.
Syntaxin-1A mutants containing point mutations (4, 11, 44) at the
a and d repeats of a predicted coiled-coil show
reduced SNAP-25 binding, supporting the involvement of this region in the interaction with SNAP-25 (Fig. 1B). Peptides
corresponding to the predicted coiled-coil-forming region of
syntaxin-1A have also been shown to inhibit neurotransmitter release
(11, 42, 44). Given these considerations, the selected 4-heptad
synthetic peptide corresponding to human syntaxin spanned from residue
191 to 218 (Fig. 1A, Peptide ST).
Design of the SB Peptide--
The region of synaptobrevin-2
between positions 27 and 96 interacts with both SNAP-25 and syntaxin-1A
in the core complex (3, 28). Synaptobrevin-2 contains a conserved
domain between residues 57 and 88, with high propensity (95%) to form
coiled-coils, and two distinct subdomains (28-42 and 52-72). Deletion
of the region spanning from residue 41 to 50 abolishes endocytosis
(45), and mutants lacking the segments 41-50 or 51-60 do not to bind to SNAP-25 and syntaxin-1A. The mutants with deletion of segment 31-38
show weak binding to t-SNAREs, whereas the deletion of segments 61-70
or 71-80 results in poor binding to syntaxin while maintaining the
interactions with SNAP-25 (47). Moreover, a single mutation (M46A)
inhibits endocytosis by 80% and reduces binding to syntaxin-1A and
SNAP-25 (45, 46). Taken together, this information suggests that the
region delimited by positions 40 and 60 is involved in the ternary
interactions that result in the assembly of the docking complex;
therefore, the peptide synthesized encompassed 4 heptads from position
40 (Fig. 1A, Peptide ST).
Design of the SNRD Control Peptide--
A control
peptide corresponding to the scrambled sequence of the selected SNAP-25
peptide was also synthesized. Randomized sequences were generated,
their secondary structures were predicted using the SOPMA method, and
the sequences with an
Secondary structure predictions using the SOPMA method (21, 22)
indicate that all three peptides may form stable Trimeric Coiled-coil Model
Theoretical Considerations--
A model of one of the trimeric
conformers of the selected peptides forming a coiled-coil structure is
shown in Fig. 1B. Given the helical wheel representation,
residues at the a- and d-positions stabilize the
structure by hydrophobic interchain interactions. According to this
model, the synaptobrevin-2 residue Met45, which upon
mutation inhibits endocytosis, would be located in the hydrophobic
core, where such a change would be predictably disruptive. The
mutations in syntaxin that reduce its binding to SNAP-25 would also be
located in positions a and d (residues denoted
with an asterisk in Fig. 1B). Interestingly, the
cleavage sites for five of the seven BoNT serotypes (BoNT A, B, D, E,
and F) are found in the model peptides, and all are in surface
locations potentially accessible to the BoNT proteases.
Interchain interactions of e- and g-positions
mediated by charged residues also contribute to the stability of a
coiled-coil (64). There are 12 charged residues in positions
e and g; accordingly, inter- or intrahelical
ionic interactions could synergistically contribute to the stability of
the coil (Fig. 1C). Polar residues implanted in the
hydrophobic core are potentially disruptive, although strategic
placement can facilitate correct oligomerization arrangements (65). In
the model, the core contains only two charged residues:
Arg198 (peptide ST) could establish an intramolecular salt
bridge with either Glu194 or Glu201 (Fig.
1B), and Arg56 (peptide SB) would be at a
suitable distance to interact with the glutamate residues in position
g of the ST peptide and form an intermolecular linkage (Fig.
1B).
In the outer layer (positions b, c, and
f), 14 negatively and 3 positively charged residues would be
exposed. This arrangement of negative charges mostly in the surface is
consistent with observations by Regazzi et al. (47) that
substitutions of negatively charged residues of synaptobrevin-2 do not
alter function (66) (Fig. 1B).
Circular Dichroism Results--
In aqueous media, all peptides
(alone or in mixtures) were unstructured, and neither increasing
peptide concentration nor changing pH, ionic strength, or divalent
cation concentration increased the
In the presence of increasing concentrations of TFE (Fig.
2, A-C), there was a
significant increase in the
Equimolar ternary mixtures SN/SB/ST in aqueous solution showed no
interaction between the non-
The 31% net increase in helicity observed in the experimental ternary
mixture spectrum with respect to the prediction could arise from the
occurrence of distinct binary complexes in the mixture. Equimolar
binary mixtures SN/SB, SN/ST, and SB/ST in aqueous solution showed no
interaction between the peptides (not shown). In the presence of 30%
TFE, the SN and SB peptides did not interact in binary mixtures (Fig.
3B). The helicity of the experimental spectrum was identical
to that predicted by the noninteracting calculated spectrum (36%). In
contrast, spectra from binary mixtures SB/ST (Fig. 3C) and
SN/ST (Fig. 3D) indicated that both pairs of peptides
interact under these experimental conditions. In each case, the
Increasingly higher peptide concentration in equimolar mixtures of the
SN, SB, and ST peptides in the presence of 30% TFE (Fig.
4A) also results in an
increase in helicity and therefore a stabilization of the complex.
Increasing the individual peptide concentrations from 10 to 30 µM results in an increase in helicity from 46 to 54%. It
is noticeable that the three spectra define a unique isodichroic point,
consistent with the occurrence of a single specific complex. When
equimolar ternary mixtures are exposed to higher concentrations of NaCl
in the presence of 30% TFE (Fig. 4B), there is a remarkable
increase in the helicity (from 55% at 0.1 M NaCl to 72%
at 0.5 M and 81% at 1 M). This feature is
consistent with hydrophobic peptide-peptide interactions as suggested
by the model (Fig. 1B). The increased
Whereas the spectra of all three peptides were independent of the
peptide concentration in aqueous solution (Fig.
5, A-C), in the presence of
30% TFE the increase in peptide concentration resulted in a
concomitant increase in the
Higher peptide concentrations induced a moderate increase in the ratio
between the peaks at 222 and 208 nm
(
The ratio of the 222- to the 208-nm peak is an operational index to
detect the presence of pure coiled-coils. For peptides stabilized at
low TFE concentrations, an equilibrium between monomeric and multimeric
states is anticipated, resulting in a profile intermediate between a
pure coiled-coil and a predominantly monomeric situation. Given that
the peptides exhibit a relatively low
To address the question of whether the increase in
The specificity of the interactions observed in binary mixtures was
tested using mixtures of the SN, SB, and ST peptides with the
SNRD control peptide (Fig.
6). The SNRD peptide was
unstructured in aqueous solution, and in the presence of 30% TFE its
Conclusion
Our study identifies a minimal entity that opens a new perspective
for the study of the molecular interactions between SNAP-25, synaptobrevin, and syntaxin. Three distinct synthetic peptides patterned after the sequences of the putative coiled-coil-forming domains of the main components of the docking and fusion complex self-assemble into a complex that exhibits spectral characteristics consistent with a coiled-coil structure. A synthetic coiled-coil ternary complex provides a basis for further developments: 1) the
ternary complex appears suitable for both crystallization and NMR
spectroscopy that, in due turn, may yield a high resolution structure
of the fusion core complex; 2) it represents a conceptual framework to
assist in the design and test of new peptide inhibitors of
neurotransmitter release; 3) it provides leads for the design of small
molecule peptidomimetic drugs; and 4) it may be valuable to generate
specific antibodies to block neurotransmitter release. Indeed, the
synthetic peptides that, as shown here, participate in the assembly of
the ternay complex in fact mimic the action of Clostridial
neurotoxins (40, 43). Therefore, our findings may lead to the
development of peptide-based agents that may be used as potential
therapy in spastic neuromuscular disorders, substituting or
complementing the current treatment with BoNTs.
We thank Professor Murray Goodman and Dr.
Joseph Taulane for the use of the Cary 61 CD spectrometer and Dr. Susan
S. Taylor for the use of the AVIV 202 CD spectrometer.
After submission of this manuscript, two
papers reported the structure of the SNARE complex as a parallel
four-helix bundle determined by x-ray crystallography (Sutton R. B.,
Fasshauer, D., Jahn, R., and Brunger, A. T. (1998) Nature
395, 347-353) and by electron paramagnetic resonance
spectroscopy (Poirier, M. A., Xiao, W., Macosko, J. C., Chan, C., Shin,
Y.-K., and Bennett, M. K. (1998) Nat. Struct. Biol. 5, 765-769). The results of our study are consistent with the high
resolution structure of the SNARE complex.
*
This work was supported by U.S. Army Medical Research and
Materiel Command Grants DAMD 17-93-C-3100 and DAMD 17-98-C-8040 (to
M. M.) and a grant from the Dystonia Medical Research Foundation (to
J. M. C.).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.
The abbreviations used are:
SNARE, SNAP
receptor; v-SNARE, vesicle SNARE; t-SNARE, target SNARE; BoNT, botulinum neurotoxin; SNAP-25, synaptosomal associated protein of 25 kDa; TFE, trifluoroethanol; HPLC, high pressure liquid chromatography.
Assembly of a Ternary Complex by the Predicted Minimal
Coiled-coil-forming Domains of Syntaxin, SNAP-25, and Synaptobrevin
A CIRCULAR DICHROISM STUDY*
ABSTRACT
Top
Abstract
Introduction
Procedures
Results & Discussion
References
-helical structure and displayed both homomeric and heteromeric
interactions. The interactions observed in ternary mixtures induce a
stabilization of peptide structure that is greater than that predicted
from individual binary interactions, suggesting the formation of a higher order structure compatible with the assembly of a trimeric coiled-coil.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results & Discussion
References
-helices (11-15). Secondary structure analysis shows that the periodic distribution of hydrophobic amino acids is consistent with a coiled-coil organization (2, 11, 12,
14, 15). Fluorescence energy transfer experiments (12) and electron
microscopy (15) further indicate that synaptobrevin and syntaxin are
aligned in parallel in the context of a ternary coiled-coil.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results & Discussion
References
] (deg·cm2·dmol
1) (25), and filtering of
the spectra using a fast fourier transform filter were performed using
the Microcal Origin 3.5 program. The percentage of -helical content
was estimated directly from the molar residue ellipticity at 222 nm as
described by Chen et al. (26). Percentages of secondary
structures were also estimated using the neural network-based K2
algorithm (27). To evaluate the spectral changes induced by
peptide-peptide interactions in mixtures, the noninteracting spectra
were calculated from the individual spectra using the equation,
where ci denotes the molar peptide
concentrations, ni represents the peptide lengths in
number of residues, and [
![[&thgr;]<SUB><UP>theo</UP></SUB>=∑(c<SUB>i</SUB> · n<SUB>i</SUB> · [&thgr;]<SUB>i</SUB>)/∑(c<SUB>i</SUB> · n<SUB>i</SUB>)](/content/vol273/issue51/fulltext/34214/img001.gif)
(Eq. 1)
]i values are observed mean
residue ellipticities.
RESULTS AND DISCUSSION
Top
Abstract
Introduction
Procedures
Results & Discussion
References
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Fig. 1.
Model of a minimal trimeric coiled-coil using
selected sequences from SNAP-25, synaptobrevin-2, and syntaxin-A.
A, schematic representation of the relative location of
SNAP-25, synaptobrevin-2, and syntaxin-A in the synaptic terminal
(black boxes). The white
boxes represent the segments corresponding to the synthetic
peptides SN, SB, and ST, respectively. The relative size of the
proteins and synthetic peptides are not to scale. The amino acid
sequences of the SN, SB, and ST peptides are given in the
box. B, triple helical coiled-coil model of the
segments of SNAP-25, synaptobrevin-2, and syntaxin-A represented by the
synthetic peptides SN, SB, and ST, respectively. The sequences have a
heptad pattern of residues (designated as a-g), where
a and d are usually hydrophobic and e
and g are frequently charged. Solid
arrows denote hydrophobic interactions in the core of the
complex, whereas broken arrows refer to potential
ionic interactions, as described in detail in C. Mutations
known to disrupt the interactions between proteins (*) are located in
the hydrophobic core. Mutations known not to interfere in the assembly
of the complex (#) are all located outside the hydrophobic core. Most
of the charged residues are located in the outer shell of the complex
(positions b, c, and f).
Hollow arrows in A and B
show the relative location of Botulinum neurotoxin cleavage sites,
which are all accessible on the surface of the complex. C,
potential interactions between charged residues of peptides SN, SB, and
ST. Charged residues in positions e and g or at
i + 3- or i + 4-positions can form intramolecular
(squares) or intermolecular (circles) salt
bridges that contribute to the overall stability of the trimeric
complex.
-helical content similar to the original
sequence were run against the Prosite data base. A peptide with the
same functional sites but without the heptad periodicity was
synthesized (sequence shown under "Experimental Procedures"), and
it was shown to be pharmacologically inactive. The SNRD
peptide, at variance to SN, did not affect
Ca2+-dependent release in chromaffin cells.
-helices in the
context of a whole protein structure. Predicted helicities for SN, SB,
and ST peptides were 62, 96, and 86%, respectively, when considered
integrated in the protein, in contrast to 42, 57, and 67% as isolated
peptides. The behavior of the isolated peptides in an aqueous
environment was predicted by using the AGADIR algorithm (23). This
program uses statistical mechanics to consider short range interactions
between residues at different pH and temperature. Given the size of our
peptides, they are predicted to be unstructured (4% for SN, 3% for
SB, and 2% for ST peptide, respectively) under aqueous conditions.
Thus, considering both sets of data, we infer that these peptides can
adopt -helical structures only in the context of the intact protein
and, therefore, that the presence of helicity-inducing conditions may
be necessary to mimic the secondary structure of the peptides in the
cytosol. TFE, a hydrophilic and hydrogen-bonding solvent, has been
widely used to stabilize marginally stable -helical structures in
potentially -helical peptides (48, 50-53). TFE is not limited to
promoting helix formation, since it has also been shown to stabilize
-turns and even -strands (54, 55). Notwithstanding, TFE-induced -helical conformation in fragments of proteins known to be -sheet in the native context has been documented (56-63). Therefore, caution must be exercised in inferring structure from CD data of peptides in
the presence of TFE, particularly with regard to the extent that it
represents the native structure in the context of the intact protein
from which the peptide sequences were selected.
-helical content. Typical
single-stranded polypeptides generally do not form stable -helices
in aqueous solution and require the additional stabilization of less
polar solvents (67, 68); therefore, we resorted to the use of the
helix-promoting solvent TFE.
-helical content. The minimal
concentration at which the peptides underwent a transition from mostly
unstructured to partially structured was approximately 30%. At that
concentration, the -helical contents of the SNAP-25, synaptobrevin,
and syntaxin peptides were 31, 44, and 32%, respectively. At the
maximum concentration of TFE used (75%), the -helical contents of
the peptides were 59, 87, and 85%, respectively. TFE increases the
-helical content, while it disrupts tertiary and quaternary
structures stabilized by hydrophobic interactions (69); therefore, it
was imperative to use a concentration of TFE low enough to marginally
stabilize the secondary structure of monomeric peptides while still
allowing the expression of tertiary interactions. Notwithstanding the
disrupting effects of TFE on the tertiary structure of oligomeric
complexes, peptide-peptide interactions producing stable oligomers have
been documented at concentrations of TFE as high as 50% (70).
Interestingly, the TFE concentration used in our experiments (30%) has
been reported to yield for numerous peptides secondary structures that
compare favorably with those of the native systems (71-73).
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Fig. 2.
CD spectra of the synthetic peptides at
different TFE concentrations. A, SN peptide;
B, SB peptide; C, ST peptide. Spectra were
recorded in 10 mM sodium phosphate, pH 7.4, 100 mM NaCl, in the presence of increasing concentrations of
TFE. Peptide concentration was 30 µM. TFE concentrations
used were 10, 20, 30 (*), 40, 50, 65, and 75%.
-helical peptides (not shown). In the
presence of TFE, the spectrum of the SN/SB/ST mixture (Fig. 3E, solid
line) was significantly different from a noninteracting spectrum (Fig. 3E, dashed line)
calculated from the three individual CD spectra (Fig. 3A).
The expected -helicity from the calculated spectrum was 35%,
whereas the -helicity from the experimental spectrum was 46%;
i.e. a 31% net increase over the predicted value. The
ratios between the intensities of the bands at 222 and 208 nm were 0.76 for the calculated and 0.80 for the experimental spectrum,
respectively. This larger 
222/208 ratio
is consistent with an increase in coiling.
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Fig. 3.
CD spectra of binary and ternary mixtures of
synthetic peptides SN, SB, and ST. Experimental (solid
line) and calculated (dashed line)
noninteracting spectra are shown for each mixture as well as recordings
corresponding to the same peptide mixture after a 24-h incubation at
5 °C. A, individual spectra of SN, SB, and ST peptides
used to determine the calculated noninteracting spectra; B,
SN/SB mixture; C, SB/ST mixture; D, SN/ST
mixture; E, SN/SB/ST ternary mixture; F,
comparison between the spectrum of the ternary mixture after a 24-h
incubation and the spectrum calculated from the summation of the binary
spectra. Peptide concentration was 30 µM for each
peptide. Spectra were recorded in 10 mM sodium phosphate,
pH 7.4, 100 mM NaCl, with 30% TFE.
-helical content calculated from the experimental spectra was
~15% greater than expected for a noninteracting mixture. Predicted helicities were 34 and 33%, respectively, for the SB/ST and SN/ST mixtures, whereas the experimental values were 39 and 38%,
i.e. 15% higher than expected for noninteracting mixtures.
-helical content with increasing ionic strength is in accordance with data for coiled-coil peptides and can be explained by the increased strength of
the hydrophobic interactions as the polarity of the medium is increased
(69).
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Fig. 4.
CD spectra of ternary mixtures of the SN, SB,
and ST peptides as function of peptide concentration and ionic
strength. A, CD spectra of equimolar mixtures of SN,
SB, and ST peptides, at individual peptide concentrations of 10, 20, and 30 µM. Spectra were recorded in 10 mM
sodium phosphate, pH 7.4, 100 mM NaCl, with 30% TFE;
B, effect of increasingly higher concentrations of NaCl on
the secondary structure of equimolar ternary mixtures of SN, SB, and ST
peptides at individual peptide concentrations of 30 µM.
Spectra were recorded in 10 mM sodium phosphate, pH 7.4, 30% TFE. NaCl concentrations used were 0.1, 0.5, and 1 M.
-helical content, as indicated by the
increase in negative ellipticity at 222 nm (Fig. 5, D-F).
Helicity increased from 30 to 53% for SN, from 41 to 49% for SB, and
from 35 to 51% for ST. This is consistent with the fact that peptides
with -helical structures that are dependent on dimerization or
oligomerization show an augmentation of -helical content as the
peptide concentration is increased (74). This presumably arises because
the equilibrium between monomeric peptide (in the form of random coil)
and coiled-coil dimer is shifted toward the formation of the
coiled-coil dimer, which increases the -helical content of the
peptide (65).
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Fig. 5.
CD spectra of the synthetic peptides at
increasingly higher concentrations in the absence or presence of
TFE. A, SN peptide; B, SB peptide; and
C, ST peptide in aqueous solution. D, SN peptide;
E, SB peptide; and F, ST peptide in 30% TFE.
Peptide concentrations were 30, 60, and 90 µM,
respectively. Spectra were recorded in 10 mM sodium
phosphate, pH 7.4, 100 mM NaCl, with or without 30%
TFE.
222/208): from 0.68 to 0.78 for SN; from
0.75 to 0.77 for SB; and from 0.88 to 0.94 for ST. The ratio between
the intensities of the bands at 222 and 208 nm may be regarded as a
measure for the extent of coiling of -helices around each other. The
222-nm CD band is mainly responsive to the -helical content, whereas
the band at 208 nm is sensitive to whether the -helix is monomeric
or is involved in tertiary contacts with other -helices (75-77).
Therefore, this is an additional criterion for the formation of stable
coiled-coil structures. Each set of curves defined a unique isodichroic
point, consistent with a single monomer-dimer equilibrium, which
indicated that the oligomerization observed was sequence-specific and
presumably stabilized by a concerted set of ion pairs in a defined
spatial arrangement.
-helical content and that
there is a substantial fraction of peptide in monomeric form, the
formation of homomeric or heteromeric arrays arising from interhelical
interactions would result in an -helical content of the mixtures
larger than that expected from a spectrum calculated from the
individual spectra (Fig. 3A).
-helicity
observed in the ternary mixture could be accounted for by mere binary
interactions between the SN, SB, and ST peptides, the calculated spectrum predicted from the sum of SN/ST and SB/ST interactions was
calculated (Fig. 3F, dashed line). The
experimental spectrum obtained from the ternary mixture (Fig.
3F, solid line) showed an -helical
content (46%) considerably higher than expected from a mixture of
dimers (38%), suggesting the presence of ternary or higher order interactions.
-helical content was similar to that of the SN peptide (spectrum not
shown). Equimolar mixtures of SNRD and SN
(SN/SNRD) showed no difference between the spectrum
calculated for a noninteracting mixture and the experimental spectrum
(Fig. 6A). Similarly, spectra obtained from
SB/SNRD and ST/SNRD binary mixtures showed no
difference with respect to the calculated noninteracting spectra (Fig.
6, B and C). The slight differences observed in
the figure are not statistically significant as assessed using a
Student's t test on the nonfiltered spectra. In the case of
mixtures assayed in the presence of 30% TFE, the situation is the
same: there is no interaction between SN, SB, or ST and the control
peptide (Fig. 6, D-F). This indicates that the interactions
observed upon increasing peptide concentration or mixing with other
peptides, either in binary or ternary mixtures, is
sequence-specific.
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Fig. 6.
CD spectra of binary mixtures of synthetic
peptides SN, SB, and ST and the control peptide SNRD.
A, SN/SNRD mixture; B,
SB/SNRD mixture; and C, ST/SNRD
mixture in aqueous solution. D, SN/SNRD mixture;
E, SB/SNRD; and F,
SB/SNRD mixture in 30% TFE. Experimental (solid
line) and calculated (dashed line)
noninteracting spectra are shown for each binary mixture. Peptide
concentration was 30 µM. Spectra were recorded in 10 mM sodium phosphate, pH 7.4, 100 mM NaCl, with
or without 30% TFE.
ACKNOWLEDGEMENTS
Note Added in Proof
FOOTNOTES
To whom correspondence should be addressed: Dept. of Biology,
University of California San Diego, 9500 Gilman Dr., La Jolla, CA
92093-0366. Tel./Fax: 619-534-0931; E-mail:
montal{at}biomail.ucsd.edu.
REFERENCES
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Abstract
Introduction
Procedures
Results & Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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