Sequence Dependence of the Folding of Collagen-like Peptides
SINGLE AMINO ACIDS AFFECT THE RATE OF TRIPLE-HELIX
NUCLEATION*
Michael S.
Ackerman
§,
Manjiri
Bhate
§,
Nigel
Shenoy
,
Konrad
Beck
,
John A. M.
Ramshaw¶, and
Barbara
Brodsky
From the
Department of Biochemistry, Robert Wood
Johnson Medical School, University of Medicine and Dentistry of New
Jersey, Piscataway, New Jersey 08854 and the ¶ Commonwealth
Scientific and Industrial Research Organization Division of Molecular
Science, Parkville, Victoria 3052, Australia
 |
ABSTRACT |
The refolding of thermally denatured model
collagen-like peptides was studied for a set of 21 guest triplets
embedded in a common host framework:
acetyl-(Gly-Pro-Hyp)3-Gly-Xaa-Yaa-(Gly-Pro-Hyp)4-Gly-Gly-amide. The results show a strong dependence of the folding rate on the identity of the guest Gly-Xaa-Yaa triplet, with the half-times for
refolding varying from 6 to 110 min (concentration = 1 mg/ml). All
triplets of the form Gly-Xaa-Hyp promoted rapid folding, with the rate
only marginally dependent on the residue in the Xaa position. In
contrast, triplets of the form Gly-Pro-Yaa and Gly-Xaa-Yaa were slower
and showed a wide range of half-times, varying with the identity of the
residues in the triplet. At low concentrations, the folding can be
described by third-order kinetics, suggesting nucleation is
rate-limiting. Data on the relative nucleation ability of different
Gly-Xaa-Yaa triplets support the favorable nature of imino acids, the
importance of hydroxyproline, the varying effects of the same residue
in the Xaa position versus the Yaa position, and the
difficulties encountered when leucine or aspartic acid are in the Yaa
position. Information on the relative propensities of different
tripeptide sequences to promote nucleation of the triple-helix in
peptides will aid in identification of nucleation sites in collagen sequences.
 |
INTRODUCTION |
The collagen triple helix is the basic structural motif found in
all fibril-forming collagens as well as some host-defense proteins such
as C1q, mannose-binding protein, and macrophage scavenger receptor (1,
2). The triple-helix conformation consists of three extended
polyproline II-like chains supercoiled around each other as determined
by x-ray fiber diffraction, crystallography, and NMR (3-7). The three
chains are staggered by one residue with respect to each other and
stabilized by interchain hydrogen bonding (5, 8, 9). This conformation
requires that every third residue must be a glycine, generating a
repeating (Gly-Xaa-Yaa)n pattern, and that a high proportion of
residues are the imino acids proline and hydroxyproline. Gly-Pro-Hyp is
indeed the most common and stabilizing tripeptide found in
collagens.1
The folding of the collagen triple helix in vivo is a
multistep process involving chain association, registration,
nucleation, and propagation (10-12). There is also evidence for the
involvement of chaperones (13, 14). Fibril-forming collagens are
synthesized at the rough endoplasmic reticulum membrane in a precursor
form, procollagen, containing both N- and C-terminal propeptides
terminating the long central triple helix. Proper chain selection and
registration is initiated by the association of the C-propeptide
domains into trimers followed by nucleation of the correctly aligned
triple helix (15, 16) and propagation in a C- to N-terminal direction (17). In the unfolded state, most proline residues in the Yaa position
of Gly-Xaa-Yaa triplets are enzymatically hydroxylated, and the
resulting hydroxyproline (Hyp) residues are required for the formation
and stabilization of the triple helix (18).
Folding studies on mature collagens are complicated by their length and
varied sequences; these complicating features can be reduced by the use
of natural or synthetic peptides. Collagen fragments have been used to
better define the folding process, e.g. the observation of third-order
kinetics for a 36-residue cyanogen bromide fragment of collagen type I
(19). Synthetic peptide models of the triple helix allow the sequence
dependence of folding to be investigated systematically by varying both
the design and the composition of the Gly-Xaa-Yaa sequences.
Investigations on synthetic model peptides, such as
(Pro-Pro-Gly)n, which adopt a stable triple-helical structure,
have allowed quantitation of third-order rate constants, the effect of
length and the testing of sophisticated theoretical models including
folding intermediates resulting from incorrectly staggered chains (20,
21). Here we present data on the refolding of a set of homologous
peptides that contain one variable Gly-Xaa-Yaa guest triplet embedded
in a Gly-Pro-Hyp-rich host sequence. This design allows the analysis of
the effect of a single triplet sequence on different triple-helix properties. Work in our laboratory has shown that all host-guest peptides analyzed so far form stable triple helices, with melting temperatures dependent on the identity of the guest triplet (22-24). This study reports folding rates for a set of host-guest peptides that
provide information on the relative propensities of different tripeptide sequences to promote nucleation of the triple helix.
 |
EXPERIMENTAL PROCEDURES |
Peptide Synthesis--
Peptides were synthesized on an Applied
BioSystems 430A synthesizer using the standard FastMoc (Applied
BioSystems) method on a N-(9-fluorenyl)methoxycarbonyl
(Fmoc)-RINK resin as described previously (22, 23). Peptides
were purified to >90% purity by reversed-phase high performance
liquid chromatography on a C-18 column eluted with a binary 10-30%
(v/v) acetonitrile/water gradient containing 0.1% trifluoroacetic
acid. Peptide identity was confirmed by laser desorption mass
spectrometry and amino acid analysis.
Sample Preparation--
Peptides were placed in vacuo
over P2O5 for more than 48 h before
weighing, dissolved in phosphate-buffered saline (10 mM sodium phosphate, 150 mM NaCl, pH 7.0), and stored at
4 °C.
Refolding Experiments--
Measurements were performed on an
AVIV Model 62DS CD2
spectrophotometer equipped with a thermoelectric temperature control in
1-mm-path length quartz cells. Peptides were denatured in glass test
tubes at 70 °C for 20 min and then rapidly cooled to 15 °C, unless otherwise specified, by quenching in an ice-water bath before
transfer into the cuvette, kept at 15 °C. Quenching time was ~5 s,
and the time needed for sample transfer was ~15 s. The ellipticity at
225 nm was monitored as a function of time over at least 30 min and
until the fraction of folded peptides exceeded 0.5 (see below), with
data intervals and averaging times of 1 to 60 s, depending on
concentration and folding speed. Experiments repeated for some peptides
indicated a deviation between their half-times of less than 10%.
Data Analysis--
The fraction of folded peptide (F)
is defined as
|
(Eq. 1)
|
where
obs,
t, and
m
represent the observed, the triple helix, and monomer ellipticity,
respectively.
t was measured directly before denaturation at
the temperature used for refolding.
m was determined by
extrapolating the initial data points to time zero. This
m
value was slightly lower than that resulting from linear extrapolation
of the monomer ellipticity observed in the high temperature region of
equilibrium melting curves, but the use of either value gave similar
results. To compare the data of different peptides independent of the
folding mechanism, the time (t1/2) at
which F = 0.5 was determined.
The concentration of monomer [A] at any given time was
calculated as
|
(Eq. 2)
|
with [A]0 denoting the initial monomeric peptide
concentration, which was assumed to be equal to the total peptide
concentration. Data were fitted to a single-step first (Eq. 3)-, second
(Eq. 4)-, or third (Eq. 5)-order kinetics:
|
(Eq. 3)
|
|
(Eq. 4)
|
|
(Eq. 5)
|
Rate constants ki were calculated after
linearization from the slope resulting from linear least squares fit.
Curves were categorized as of ith order based on maximum
linear correlation coefficients.
 |
RESULTS |
Peptide Design and Stability--
Host-guest peptides of the form
acetyl-(Gly-Pro-Hyp)3-Gly-Xaa-Yaa-(Gly-Pro-Hyp)4-Gly-Gly-amide
provide a useful template to evaluate the contribution of individual
Gly-Xaa-Yaa triplets to triple-helix properties (22-24). To assure
formation of a stable triple-helix, the guest triplet is flanked by
stabilizing Gly-Pro-Hyp triplets. The N and C termini are blocked by
acetylation and amidation, respectively, to ensure that the only
ionizable groups, if any, would be those introduced in the guest
triplet and to eliminate charge repulsion at the ends of the triple
helix. The peptide length is designed to be short enough so that the
effects of a single guest triplet would not be masked by the constant
part of the structure but long enough to ensure triple-helix stability (22). Because imino acids are found at high frequency in triple helices, guest triplets of the form Gly-Xaa-Hyp, Gly-Pro-Yaa, and
Gly-Xaa-Yaa were considered, with the Xaa and Yaa residues occupied by
the most common nonpolar residues, and charged residues found in
collagens. In the following, we refer to the different peptides by
their guest triplet sequence. This design allows analysis of the
effects on folding of a single residue within a defined triple-helical environment.
CD measurements of all host-guest peptides indicate triple-helical
structures at low temperature. The spectra show a characteristic maximum near 225 nm with a mean residue ellipticity in the order of
4,000 deg cm2 dmol
1, which decreases upon
unfolding (Fig. 1, inset).
Equilibrium unfolding curves exhibit a highly cooperative behavior with
melting temperatures ranging from 20 to 45 °C, depending on the
identity of the guest triplet (22-25). The curves can be fitted to a
two-state trimer to monomer transition, an assumption supported by
analytical ultracentrifugation experiments performed on closely related
peptides (26).

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Fig. 1.
Refolding curve of host-guest peptide
Gly-Ala-Hyp. The refolding of peptide Gly-Ala-Hyp
(concentration = 1 mg/ml) was monitored by following the recovery
of ellipticity at 225 nm at 15 °C after denaturation at 70 °C for
20 min The signal was normalized to the fraction of folded peptide
according to Equation 1. The time t1/2 (~ 8.3 min)
at which half the molecules are folded (F = 0.5) is
indicated by a dashed line. The inset shows the
CD spectra of the same sample recorded at 15 °C ( )
and 70 °C (- - -). The arrow marks the transition
followed in the refolding experiment.
|
|
Folding of Host-Guest Peptides--
Refolding rates were measured
for a total of 21 host-guest peptides (concentration = 1 mg/ml).
Despite the variations in stability of the host-guest peptides, a
common folding temperature of 15 °C was selected. At this
temperature all peptides showed a fraction of folded peptide close to
one in their equilibrium melting curves, and little dependence of the
folding rate on temperature of folding was observed at 5, 10, 15, and
20 °C (data not shown). As an example, the signal recovery for
peptide Gly-Ala-Hyp at 15 °C is shown in Fig. 1, which also
illustrates the determination of t1/2 values as a common measure to compare the refolding
behavior of the different peptides. The
t1/2 values for the host-guest peptides
varied between 6 and 110 min, revealing that the folding rate
critically depends on the sequence of the guest triplet (Table
I). For example, the folding half-times of peptides Gly-Pro-Hyp, Gly-Ala-Hyp, Gly-Pro-Ala, and Gly-Ala-Ala are
6.0, 8.3, 12, and 21 min, respectively, at 15 °C (Fig.
2). The fast folding of Gly-Pro-Hyp is
decreased slightly by the substitution of Pro by Ala in the Xaa
position and somewhat more when the Hyp is replaced by an Ala.
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Table I
Sequence dependence of refolding
Refolding of the indicated peptides (concentration = 1 mg/ml)
after denaturation at 70 °C for 20 min was monitored at 15 °C,
and half-times t1/2 (in min) were determined.
Peptides are sorted in complementary sets.
|
|

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Fig. 2.
Effect of alanine in positions Xaa and Yaa on
refolding kinetics. Recovery of the CD signal at 225 nm was
monitored at 15 °C after 20 min denaturation at 70 °C, and the
signal was converted to the fraction of folded peptide for Gly-Pro-Hyp
( ), Gly-Ala-Hyp ( ), Gly-Pro-Ala ( ) and Gly-Ala-Ala ( )
(concentration = 1 mg/ml).
|
|
Guest triplets with Hyp in the Yaa position (Gly-Xaa-Hyp) fold fastest
and show only a small dependence on the identity of the residue in the
Xaa position. Gly-Pro-Yaa guest triplets with Pro in the Xaa position
show a larger range of folding rates and a stronger influence of the
identity of the Yaa residue. In the Gly-Pro-Yaa peptides, the residues
glutamine and glutamic acid have a very similar influence on the
kinetic behavior (t1/2 = 22 min for Gln
and 27 min for Glu), whereas an exchange of aspartic acid for
asparagine drastically slows down the refolding
(t1/2 = 38 min for Asn and 98 min for
Asp). The combination of nonimino acids in both the Xaa and Yaa
positions (Gly-Xaa-Yaa) leads to a broad distribution of
t1/2 values, with peptides Gly-Ala-Leu
and Gly-Asp-Ala folding particularly slowly. In general, leucine was
observed to decrease the folding rate compared with Ala, with the
effect being most striking in the Yaa position.
Kinetic Analysis of Triple-helix Folding--
To understand the
influence of the single guest triplet on the folding mechanism, the
folding data at 1 mg/ml was analyzed by fitting to first-, second-, and
third-order kinetics (Equations 3-5). Considering the full folding
process, some peptides showed best fits when assuming a first-order
kinetics, whereas others showed best fits when either second- or
third-order kinetics were assumed. Analyses of the folding curves of
several peptides in which only the late data points were fitted to
third-order kinetics (Equation 5) revealed that these regions show a
reasonably straight line in
1/[A]02(t) plots (data not shown).
This suggests that at a sufficiently low monomer concentration, the
third-order step, which relates to chain association and/or nucleation,
becomes the rate-limiting step for trimer formation.
Folding via a third-order kinetic process should show a profound
dependence on concentration, whereas the first-order kinetics of
propagation should be independent of concentration. Measurements of the
kinetics of folding for a set of 8 peptides at a concentration of 0.4 mg/ml (Fig. 3A) indicated that
at this lower concentration all but the very early time points (t < 10 to 300 s) followed a straight line when plotted as
1/[A]02 (t), giving a good fit to
third-order kinetics (Fig. 3B). This indicates that at a
sufficiently low monomer concentration, chain association/nucleation
becomes the rate-limiting kinetic step in triple-helix folding. The
apparent third-order rate constants derived from the slopes varied from
470 to 16,000 M
2 s
1, with S.D.
of approximately ± 10%. The lowest third-order rate constants
were found for peptides Gly-Ala-Leu and Gly-Pro-Asp, and the largest
was found for Gly-Pro-Hyp (Table II). The
Gly-Pro-Pro peptide had a slower folding rate than Gly-Pro-Hyp, showing
the effect of a hydroxyl group at one position. The large variation in
the rate constants suggests that for these relatively short peptides,
the chain association/nucleation step critically depends on the
identity of the single guest triplet, and at this low concentration, any other processes like helix propagation have only a marginal contribution.

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Fig. 3.
Refolding kinetics of host-guest peptides at
low concentration. A, recovery of the CD signal at 225 nm was monitored at 15 °C after denaturation, and the signal was
converted to the fraction of folded peptide at a concentration of 0.4 mg/ml for (from top to bottom) Gly-Pro-Hyp, Gly-Pro-Pro, Gly-Ala-Hyp,
Gly-Pro-Ala, Gly-Leu-Ala, Gly-Ala-Ala, Gly-Ala-Leu, and Gly-Pro-Asp.
B, curves linearized according to Equation 5 are shown for
the same peptide set. Third-order rate constants (Table II) were
derived from the slope of the fitted lines.
|
|
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Table II
Effect of sequence on third-order rate constants
Refolding of the indicated peptides (concentration = 0.4 mg/ml)
after denaturation at 70 °C for 20 min was monitored at 15 °C.
Third-order rate constants k3 were determined
according to Equation 5 as outlined in Fig. 4B.
|
|
 |
DISCUSSION |
The folding of collagen molecules in vivo is an
intricate and coordinated process (10, 12, 27). The formation of a
trimer requires association and registration of three chains, which is mediated for fibril-forming collagens by the globular C-terminal propeptide domain (10-12) (Fig. 4).
C-propeptide trimerization constrains the three chains at the C
terminus such that nucleation can occur at the C terminus of the
triple-helical region. Nucleation is considered to be the process in
which a series of tripeptide units from the three chains adopt
appropriate collagen-like
,
angles and form interchain hydrogen
bonds. At the C termini of fibril-forming collagens are 5-6 sequential
triplets of the form Gly-Xaa-Hyp (often Gly-Pro-Hyp units), which are
thought to constitute all or part of the nucleation site. For example,
in collagen type III, C-terminal Gly-Xaa-Hyp triplets are required for
nucleation, and deletion experiments showed that as long as two
Gly-X-Hyp triplets are retained, nucleation is effective, and
triple-helix folding is complete (16). Following nucleation,
propagation proceeds in a zipper-like manner from the C to N terminus.
The rate-limiting step of propagation is cis-trans
isomerization at imino acid peptide bonds, which have a significant
proportion of cis bonds in unfolded chains (17, 28, 29).

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Fig. 4.
Schematic of triple-helix folding.
A, folding of collagen, showing association of C-propeptides
followed by nucleation of three chains at the C terminus and then
propagation. B, folding of host-guest peptides, showing
association and nucleation of three nonlinked peptide chains.
Nucleation is shown at the C terminus, but it is likely that it can
occur at any site within the peptide. The guest triplet is indicated by
a bold line.
|
|
The studies reported here on a set of host-guest peptides demonstrate
that the folding rate of the triple helix critically depends on the
sequence of a single guest triplet. Analysis of our data indicates that
the folding proceeds via a mechanism involving more than a single
reaction step and that the folding involves a third-order process that
becomes rate-limiting at low concentrations. The steps requiring the
involvement of three polypeptide chains are chain association and
nucleation. Although the association process, which is limited by
diffusion, is unlikely to be significantly affected by sequence,
triple-helix nucleation is known to be facilitated by the presence of
conformationally restricted imino acids and is thus expected to be
sequence-dependent (30) (Fig. 4). In the present study, the
magnitude of the third-order rate constant is strongly affected by the
identity of the guest triplet and is greater for imino acid-containing
triplets, suggesting it is the nucleation step itself reflected by
these values. NMR studies on peptides with specific 15N
labels indicated that nucleation can occur at (Gly-Pro-Hyp)n sites at either end of a peptide (31). For the Gly-Pro-Hyp-enriched host-guest peptides, it is realistic to assume that nucleation could
begin at any tripeptide unit in the chain (32) (Fig. 4). Previous
findings suggest the nucleation domain is as long as six triplets in
noncovalently linked peptides (20, 21), making this a dominant event in
short peptides.
The third-order rate constants and half-times of folding yield
information concerning the relative propensity of different residues in
the Xaa and Yaa position to initiate triple-helix nucleation. Entropic
factors are likely to play an important role in nucleation because
imino acids are sterically constrained to dihedral angles similar to
those found in collagen. Gly-Pro-Hyp is the fastest folding triplet,
and all Gly-Xaa-Hyp triplets are very favorable. The Hyp residue
appears more favorable than Pro, as seen in the faster folding rate of
Gly-Pro-Hyp versus Gly-Pro-Pro. It has been suggested that
the OH of Hyp has an inductive effect, leading to a decrease in the
cis:trans isomer ratio compared with Pro in the
unfolded state (33). A decrease in the cis isomer concentration could accelerate the propagation step. In addition, the
decreased cis:trans ratio could promote
nucleation by making it more likely to find a stretch of contiguous
all-trans tripeptide units and by creating a more rigid
monomer chain (34). Although Pro in the Xaa position also can lead to
favorable folding, the identity of the nonimino acid residue in the Yaa
position of Gly-Pro-Yaa triplets has a very strong influence. For
example, Gly-Pro-Ala is a fast folding peptide, whereas Gly-Pro-Asp has
the slowest folding rate observed.
In addition to entropic factors, the influence of specific side chains
in promoting nucleation may relate to steric factors, electrostatic
interactions, and hydrogen bonding. The difficulty in packing bulky
residues such as Leu in the Yaa position (35), which is less exposed
than the Xaa position, may contribute to the slow folding of
Gly-Pro-Leu and Gly-Ala-Leu peptides. Despite its large side chain,
arginine in the Yaa position is favorable in promoting chain nucleation
as well as for stabilization (24), and both features may be related to
its ability to form multiple hydrogen bonds combined with its
restricted mobility (36, 37). Gly-Pro-Asp is the slowest folding
peptide, suggesting an unfavorable effect of aspartic acid in the Y
position. It was previously observed that aspartic acid in the Yaa
position had a destabilizing effect on the triple helix. Both the
decreased folding rate and low stability may be related to the
restricted rotational freedom of Asp in the triple helix, hindering its
participation in interchain hydrogen bond formation (23).
The systematic exchange of single guest triplets embedded in an
otherwise constant environment allows their influence on folding to be
related to their contribution to triple-helix stability. The
relationship between folding half-times and the melting temperatures of
the host-guest peptide concentrations set at 1 mg/ml was considered (Fig. 5). All Gly-Xaa-Hyp peptides have
fast folding rates and high stabilities, with a small range for both
t1/2 values and melting temperature values. The
Gly-Pro-Yaa peptides show a broad range for both folding half-times
(12-98 min) and melting temperatures (30-45 °C), with the more
stable peptides tending to fold faster. For Gly-Xaa-Yaa triplets with
no imino acids, four peptides with similar thermal stabilities were
found to have very different folding times. This suggests the
interactions determining folding differ from those important for
stability for tripeptides with no imino acids.

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Fig. 5.
Relationship between refolding rate and
thermal stability. Refolding half-times
t1/2 are plotted versus
melting temperatures, both determined at 1 mg/ml. Letters
adjacent to the symbols denote the residues in the
Gly-Xaa-Yaa guest triplet (standard one letter code) within the peptide
sets Gly-Xaa-Hyp ( ), Gly-Pro-Yaa ( ), and Gly-Xaa-Yaa ( ).
Melting temperatures are from Shah et al. (22), Chan
et al. (23), and Yang et al. (24) except for
Gly-Asn-Hyp, Gly-Gln-Hyp, Gly-Pro-Asn, Gly-Pro-Gln, and Gly-Asp-Ala
(Shenoy, N., Beck, K., and Brodsky, B., unpublished data).
|
|
The host-guest peptide set shows the wide range in effectiveness of
Gly-Xaa-Yaa tripeptides in a fixed Gly-Pro-Hyp environment to
facilitate or depress nucleation. The nucleation step of peptides differs from that of collagen in that this step occurs in three independent peptides, whereas triple-helix nucleation in collagen occurs in a molecule that is linked together by the association of
disulfide-linked C-propeptides (Fig. 4). Despite this difference, it is
likely that the propensity of individual tripeptides to nucleate a
peptide triple helix can be applied to the ability of different
sequences at the C terminus of collagen to serve as a nucleation site.
In addition, it is possible that interruptions in the
(Gly-Xaa-Yaa)n-repeating sequence, as found normally in
basement membrane collagen or for osteogenesis imperfecta Gly
Xaa mutations in type I collagen, may terminate propagation (38, 39),
making a renucleation event necessary to complete triple-helix formation. Information on the propensity of different Gly-Xaa-Yaa triplets to promote nucleation will aid in the identification of such
renucleation sequences.
 |
ACKNOWLEDGEMENTS |
We are grateful to Alan Kirkpatrick for
peptide synthesis and Nick Bartone for peptide characterization. We
also thank Drs. Naina K. Shah, Wei Yang, and Ellen Doss for helpful
comments, Chirag Shah for preliminary results, and Ms. Virginia C. Chan for experimental guidance in the early stages of this project. Furthermore, we appreciate the continuous discussions with Dr. Jean Baum.
 |
FOOTNOTES |
*
This work has been supported by the National Institutes of
Health Grant AR19626 (to B. B.), a National Science Foundation U. S.-Australia International Cooperative Research grant (to B. B.), and
the Australia/U. S. A. Bilateral Science Program (to J. A. M. R.).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.
§
Equal contributors to this work.
To whom correspondence should be addressed: Dept. of
Biochemistry, Robert Wood Johnson Medical School, University of
Medicine and Dentistry of New Jersey, 675 Hoes Lane, Piscataway, NJ
08854-5636. Tel.: 732-235-4048; Fax: 732-235-4783; E-mail:
brodsky{at}rwja.umdnj.edu.
1
Standard three-letter nomenclature is used to
describe peptide sequences with Hyp representing 4-hydroxyproline. In
the standard one-letter code for amino acids, O is used to represent
4-hydroxyproline. Host-guest peptides are named by the sequence of
their guest triplet.
 |
ABBREVIATIONS |
The abbreviation used is:
CD, circular
dichroism.
 |
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