From the Department of Biophysical Chemistry, Biozentrum,
Universität Basel, CH-4056 Basel, Switzerland and the
Shriners Hospital for Children, Research Department, and
Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, Oregon 97239
Received for publication, December 19, 2002, and in revised form, January 21, 2003
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ABSTRACT |
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The directional dependence of folding rates for
rod-like macromolecules such as parallel Proteins fold along multiple pathways (1), and rod-like
macromolecules may fold in two different directions depending on the
site of nucleation. Experimental evidence concerning the directionality of kinetics is limited. An interesting study with a two-stranded coil-coil structure tethered by disulfide bridges at either the NH2 or COOH terminus has been performed (2) and for
collagen NMR diffusion was proposed as a potential method to determine directionality (3). The collagen triple helix is a linear structure composed of three left-handed polyproline-II-type helices. Chains in
this conformation are not stable as individual structures, but
associate to form a right-handed triple helix, which is stabilized by
hydrogen bonds between chains (4). Formation of the triple helix is
only possible if every third residue in the sequence of the chains is
glycine (Gly). Imino acid residues favor polyproline-II-type helices
and therefore the frequency of proline (Pro) residues in the
typical collagen like sequence (Gly-X-Y)n
is high in X and Y positions. 4-Hydroxyproline
plays a special role in stabilizing the triple helix (5, 6) and is
frequently in the Y position. Collagen triple helices are
very abundant structures. They occur in all collagens as long rod-like
elements and form short rods in the first component of complement C1q,
lung surfactant protein SPA, mannose-binding protein, scavenger
receptors, and many other extracellular proteins (7). Collagen triple
helices are slowly folding structures with peptide cis-trans
isomerization as the rate-limiting step (8). Cis-trans isomerization is
often rate-limiting also for globular proteins (9) and is dominating for collagens because of the many X-Pro bonds (X
stands for any amino acid residue) with their high probability to form
cis-peptide bonds in the unfolded state. Another peculiarity for
collagens is the need of nucleation domains for correct and
sufficiently fast triple helical folding. It was found that trimerizing
non-collagenous domains is essential for initial chain association and
chain registration. This requirement for oligomerization and
registration was explained by the need to establish a high local
concentration at the nucleus and to prevent mismatched structures. For
thermodynamic and kinetic studies, collagen-like peptides with designed
oligomerization and nucleation domains have been particularly useful
(10-13). Spontaneous folding of the collagen triple helix is extremely
slow, follows a third order reaction, and is therefore strongly
concentration dependent (11, 14). For most collagens NC-1 domains are
found at the COOH terminus, leading to the idea that triple helices may
only or preferentially fold from the COOH terminus. Recently it was
suggested that a three-stranded coiled-coil domain at the NH2 terminus of collagen XIII can induce collagen folding
from the NH2 terminus (15). Similar suggestions have been
made for other collagens (16). This stimulated us to measure the rate of collagen triple helix folding in both directions applying designed proteins in which a globular oligomerization domain or a disulfide knot
is attached to either the NH2 or COOH terminus of
(Gly-Pro-Pro)10.
Construction of Expression Plasmids and Production of Recombinant
Proteins--
The designed proteins
(GPP)10-Cys2 and (GPP)10-foldon
were expressed in Escherichia coli as described previously
(11, 17). These models were used by these authors to study the increase in stability caused by the trimerization of the fused phage protein foldon (17) or by the linkage of the three chains by the disulfide knot
Cys2 = GPPGPCCGGG of type III collagen (11). The inverted protein with the oligomerization disulfide knot at the NH2
terminus Cys2-(GPP)10 was expressed using the
same strategy but an additional Gly-Ser-spacer was inserted between
Cys2 and the (GPP)10 moiety. The protein
foldon-(GPP)10 was synthesized by
Fmoc1 chemistry with a
Ser-Gly-Ser-Gly-spacer between the domains (for exact sequences, see
the legend of Fig. 1).
Oxidative Disulfide
Cross-linking--
(GPP)10-Cys2 and
Cys2-(GPP)10 were trimerized at 20 °C by
their (GPP)10 domains, and formation of the disulfide knot
was achieved by oxidation of the fully reduced material with a mixture
of oxidized and reduced glutathione at a molar ratio of 9:1. Trimer
formation was verified by mass spectroscopy and by analytical ultracentrifugation.
Mass Spectral Analysis--
For mass spectral analysis, the
peptides were chromatographed on a 100-µm inner diameter column
packed with Vydac C18 reverse-phase material (5-µm particle size).
The proteins were eluted with a linear 20-min gradient from 0.1%
trifluoroacetic acid to 80% acetonitrile, 0.1%
trifluoroacetic acid at a flow rate of 1 µl/min. The outlet of
the column was directed to a microspray needle, which was pulled from
100-µm inner diameter 280-µm outer diameter fused silica capillaries (LC Packings) on a model P-2000 quartz micropipette puller
(Sutter Instrument Company). The needle was placed into an XYZ
micropositioner, and the voltage was applied directly to the sample
stream through the capillary union (18). Spray voltages were usually
between 1100 and 1400 V. Mass determinations were carried out on a
TSQ7000 triple quadrupole mass spectrometer (Finnigan). Formation of
trimers of the predicted mass was verified for
(GPP)10-Cys2 and
Cys2-(GPP)10. In foldon-(GPP)10 and
(GPP)10-foldon the three chains are not connected by
covalent bonds and mass spectrometry demonstrated monomers of the
predicted mass.
Analytical Ultracentrifugation--
Sedimentation equilibrium
experiments were performed on a Beckman Optima XL-A analytical
ultracentrifuge (Beckman Instruments) equipped with 12-mm Epon
double-sector cells in an An-Ti-60 rotor. The peptides were analyzed in
5 mM sodium phosphate buffer, pH 7.4, containing 150 mM NaCl. Sedimentation velocity runs were performed at a
rotor speed of 56,000 rpm, and sedimenting material was assayed by
absorbance at 234 nm. Sedimentation coefficients were corrected to
standard conditions (water, 20 °C; Ref. 19). Molecular masses were
evaluated from lnA versus r2 plots,
where A is the absorbance, and r is the distance
from the rotor center (19). A partial specific volume of 0.73 ml/g was
used for all calculations.
CD Spectroscopy--
CD experiments were acquired on a Cary 61 (Varian) or an Aviv 202 spectropolarimeter, equipped with a
thermostatted 1-mm path length quartz cell. Data were normalized for
concentration and path length to obtain the mean molar residue
ellipticity after subtraction of the buffer contribution. Thermal
stability was determined by monitoring the change in the mean molar
residue ellipticity ([ The thermal transition profile of
Cys2-(GPP)10 was measured by the change in
circular dichroism at 221 nm, and a midpoint transition temperature
Tm = 67 °C was obtained which is 15 °C lower
than the value for (GPP)10-Cys2 (11) (Fig.
1a, Table
I). As previously observed for
(GPP)10-Cys2, the thermal transition curves of
Cys2-(GPP)10 were not fully reversible at temperatures higher than 70 °C, probably because of chemical
degradation. Measurements were therefore performed in the presence of 2 M guanidine hydrochloride (GuHCl) resulting in
Tm values of 40 and 63 °C for
Cys2-(GPP)10 and
(GPP)10-Cys2, respectively (Table I). In the
presence of guanidine HCl the transition was fully reversible according
to two criteria: (i) after heating to 50 °C for
Cys2-(GPP)10 and to 70 °C for
(GPP)10-Cys2, for 5 min, the sample was cooled to 20 °C and the value before heating returned after 2 h; (ii) a second transition profile after this refolding was identical to the
initial one. The thermal transition of foldon-(GPP)10 was measured by circular dichroism at 210 nm, where the spectrum of foldon
alone shows no change upon increasing the temperature (11). The
midpoint transition temperature Tm = 53 °C
corresponds to the (GPP)10-triple helix and is 17 °C
lower than the value for the (GPP)10 domain in
(GPP)10-foldon measured by the same method (Fig.
1b). Full reversibility was demonstrated by repeated scanning. Comparing the Tm values with those of
(GPP)10-triple helix without oligomerization domains (Table
I), the results demonstrate that nucleation at either the
NH2 and COOH terminus stabilizes the triple helix. The mode
of stabilization by foldon at the COOH terminus was investigated in
some detail (11, 17), and the crystal structure of
(GPP)10-foldon has recently been solved.2 It was
concluded that the stabilization is entropic in nature such that the
oligomerization domain creates an internal concentration of about 1 M at the junction between foldon (or the disulfide knot)
and (GPP)10. A number of unfavorable interactions at the domain junction counteract the entropic stabilization. First, foldon-(GPP)10 has a different linker sequence to that in
(GPP)10-foldon. Second, there are repulsive carboxylate
charges at the COOH-terminal end of the triple helix that are not
present in (GPP)10-foldon, and this may explain the lower
transition temperature. The peptide Ac-(GPP)10-NH2 has a Tm of
45 °C in H2O as compared with 25 °C for
(PPG)10.3 Also,
for Cys2-(GPP)10 the linker sequence is
different from that in (GPP)10-Cys2, and
COOH-terminal repulsive charges at the end of the triple helix are
present, leading to a reduced stability. In all cases the internal
concentrations imposed by the oligomerization domains are expected to
be approximately identical.
-helical coiled-coils, DNA
double-helices, and collagen triple helices is largely unexplored. This
is mainly due to technical difficulties in measuring rates in different directions. Folding of collagens is nucleated by trimeric
non-collagenous domains. These are usually located at the COOH
terminus, suggesting that triple helix folding proceeds from the COOH
to the NH2 terminus. Evidence is presented here that
effective nucleation is possible at both ends of the collagen-like
peptide (Gly-Pro-Pro)10, using designed proteins in which
this peptide is fused either NH2- or COOH-terminal to a
nucleation domain, either T4-phage foldon or the disulfide knot of type
III collagen. The location of the nucleation domain influences
triple-helical stability, which might be explained by differences in
the linker sequences and the presence or absence of repulsive charges
at the carboxyl-terminal end of the triple helix. Triple helical
folding rates are found to be independent of the site of nucleation and
consistent with cis-trans isomerization being the rate-limiting step.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
]) at a fixed wavelength of 221 nm for
(GPP)10-Cys2 and
Cys2-(GPP)10 and 210 nm for
foldon-(GPP)10 and (GPP)10-foldon as a function
of temperature. Data analysis was performed with the LABView (National
Instruments) and Sigma Plot (Jandel Scientific) software packages.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Thermal stability of foldon- and disulfide
knot-containing (GPP)10. a,
temperature-induced unfolding profiles of the collagen-like peptides
Cys2-(GPP)10 (red curve) and
(GPP)10-Cys2 (blue curve) monitored
by CD following the change of the mean molar residue ellipticity at 221 nm ([ ]221) at a total chain concentration of 50 µM in 5 mM sodium phosphate buffer containing
2 M GuHCl, respectively. The amino acid sequences are
GSYGPPGPCCGSGPP(GPP)10 for
Cys2-(GPP)10 and
GS(GPP)10GPPGPCCGGG for
(GPP)10-Cys2. b, temperature-induced
unfolding profiles of foldon-(GPP)10 (red curve)
and (GPP)10-foldon (blue curve) monitored by CD
at 210 nm in 5 mM phosphate buffer, containing 150 mM NaCl. The heating rate was 10 °C/h. The amino acid
sequence of foldon-(GPP)10 is
GYIPEAPRDGQAYVRKDGEWVLLSTFLSGSG(GPP)10 and
GS(GPP)10GSGYIPEAPRDGQAYVRKDGEWVLLSTFL for
(GPP)10-foldon.
Midpoint transition temperatures Tm of the (GPP)10
domain in designed proteins and their molecular masses
The designed proteins with oligomerization domains either at the
NH2 or COOH terminus were then used to compare the rates of
refolding. It is known that nucleation of triple helices from free
(GPP)10 chains is extremely slow,
concentration-dependent, and of apparent third order (11).
Connection of three chains by oligomerization domains increases the
intrinsic chain concentrations to a level at which cis-trans
isomerization steps in helix propagation, rather than chain finding
steps, become rate-limiting (11). Given the very high concentration
dependence of nucleation, it has to be expected that helix propagation
will be preferentially initiated at the end at which the
oligomerization domain is attached, although we cannot exclude the
possibility that a small fraction of helix initiations may also occur
at other sites. Fig. 2 shows the
time-resolved phases that follow a fast and kinetically unresolved jump
of the circular dichroism signal. The fast phase mainly consists of the
temperature-dependent change of circular dichroism, which is
visible also in temperature regions where no transition takes place
(Fig. 1). It may also include a very small fast phase of triple helix
formation before the first cis-peptide bond is met (8, 20). The
slow phase reflects the major part of the transition of the
(GPP)10 domain, and long triple helices of real collagens fold entirely with a slow phase determined by cis-trans isomerization (8). For Cys2-(GPP)10 and
foldon-(GPP)10 it was verified that the kinetic time
courses did not change upon increasing the concentration by a factor of
4 at 10 or 20 °C. For (GPP)10-Cys2 this was
shown previously (11). Figs. 2 and 3
clearly demonstrate that the first order rate constant of folding is
identical for proteins carrying the oligomerization domains at opposite
ends. The refolding rate constant for
Cys2-(GPP)10 was 0.00037 s1
measured at 20 °C and a peptide concentration of 100 µM. For foldon-(GPP)10 a value of 0.00098 s
1 was determined for a peptide concentration of 51 µM at the same temperature. Also, the activation energies
obtained from an Arrhenius plot of rate constants measured at different
temperatures turned out to be identical within the limits of
experimental error (Fig. 3). Values of activation energy larger than 50 kJ/mol confirm cis-trans isomerization steps as rate-limiting events.
Deviations from the maximum activation energies predicted for this step
have been discussed (11).
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In summary the results demonstrate that collagen triple helix formation
can be nucleated at both ends. The rates are equal within experimental
errors. To exclude possible effects of the nature of the nucleation
domain, two very different oligomerization sequences were employed. It
was shown that their influence on protein stability was slightly
different and also that the location of the domains at either end of
the collagen-like domain was an important factor. These differences may
be explained by energetic differences in the linker sequences between
the collagen triple helix and the oligomerization domains and the
presence of charges at the end of the triple helix. The rate-limiting
step of cis-trans isomerization is, however, independent of the
direction of folding.
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ACKNOWLEDGEMENT |
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We thank Ariel Lustig for the analytical ultracentrifugation measurements.
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FOOTNOTES |
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* This work was supported by grants form the Schweizerische Nationalfonds (to J. E.) and from the Shriners Hospital for Children (to H. P. B.).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.
§ To whom correspondence should be addressed: Shriners Hospital for Children, 3101 SW Sam Jackson Park Rd., Portland, OR 97239. Tel.: 503-221-3433; Fax: 503-221-3451; E-mail: hpb@shcc.org.
Published, JBC Papers in Press, January 22, 2003, DOI 10.1074/jbc.C200698200
2 J. Stetefeld, S. Frank, M. Jenny, T. Schulthess, R. A. Kammerer, S. Boudko, R. Landwehr, and J. Engel, submitted for publication.
3 K. Mizuno and H. P. Bächinger, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: Fmoc, N-(9-fluorenyl)methoxycarbonyl; GuHCl, guanidine hydrochloride.
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REFERENCES |
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