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Collagen Triple Helix Formation Can Be Nucleated at Either End*

Sabine Frank, Sergei Boudko, Kazunori MizunoDagger , Therese Schulthess, Jürgen Engel, and Hans Peter BächingerDagger §

From the Department of Biophysical Chemistry, Biozentrum, Universität Basel, CH-4056 Basel, Switzerland and the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The directional dependence of folding rates for rod-like macromolecules such as parallel alpha -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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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 ([theta ]) 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
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INTRODUCTION
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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.


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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 ([Theta ]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.

                              
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Table I
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 s-1 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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Fig. 2.   Refolding rates of (GPP)10 with disulfide knots. Refolding rates were measured by circular dichroism at 221 nm. a, refolding kinetics of Cys2-(GPP)10 (red curve) and (GPP)10-Cys2 (blue curve) at 20 °C in 5 mM phosphate buffer containing 2 M GuHCl after a 5-min unfolding at 50 °C for Cys2-(GPP)10 and 70 °C for (GPP)10-Cys2. Solid lines are experimental data, and dashed lines were fitted according to Ref. 11. b, temperature dependence of the refolding rate of Cys2-(GPP)10 (red circles and line) and (GPP)10-Cys2 (blue circles and line). The activation energies are 53 kJ/mol and 52.5 kJ/mol, respectively.


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Fig. 3.   Refolding rates of (GPP)10 with foldon domains. Refolding rates were measured by circular dichroism at 210 nm. a, refolding kinetics of foldon-(GPP)10 (red curve) at 10 °C and (GPP)10-foldon (blue curve) at 7 °C in 5 mM phosphate buffer containing 150 mM NaCl after a 3-min unfolding at 70 °C. Solid lines are experimental data, and dashed lines were fitted according to Ref. 11. b, temperature dependence of the refolding rate of foldon-(GPP)10 (red circles and line) and (GPP)10-foldon (blue circles and line). The activation energies are 50.0 kJ/mol and 54.5 kJ/mol, respectively.

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.

    ACKNOWLEDGEMENT

We thank Ariel Lustig for the analytical ultracentrifugation measurements.

    FOOTNOTES

* 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.

    ABBREVIATIONS

The abbreviations used are: Fmoc, N-(9-fluorenyl)methoxycarbonyl; GuHCl, guanidine hydrochloride.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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