Ferrocene-Assisted Stabilization of Collagen Mimetic Triple Helices

Synthesis and biological applications of collagen-model triple-helical peptides ... Xavier de Hatten , Zoe Cournia , Ivan Huc , Jeremy C. Smith , Nils...
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Bioconjugate Chem. 2006, 17, 84−89

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Ferrocene-Assisted Stabilization of Collagen Mimetic Triple Helices: Solid-Phase Synthesis and Structure Subrata K. Dey and Heinz-Bernhard Kraatz* Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, Canada. Received September 5, 2005; Revised Manuscript Received November 8, 2005

A series of ferrocene-containing collagen models Fc-CO-(Pro-Hyp-Gly)n-Cys (n ) 4 (1), 6 (2), 7 (3), 8 (4), 9 (5)) were synthesized by solid-phase synthesis. Biophysical studies using circular dichroism (CD) show that these collagen analogues form triple-helical conformations, and the peptides showed a range of thermal stabilities ((Tm), 38-74 °C). Results also indicate that the ferrocene (Fc)-labeled collagen models possesses a higher triple-helical propensity than the unlabeled collagen models as demonstrated by the higher melting temperatures and thermodynamic parameters, and we conclude that the Fc group at the N-terminal position of the peptide strands increases the stability of the triple helix.

INTRODUCTION

Scheme 1

The design of collagens with a high melting temperature (Tm) is a formidable task, which has attracted significant attention over the past few years. It has been well-known that the collagen molecule consists of three polypeptide chains; each undergoes a transition to a single coil state as the temperature increases (1). The thermal stability of the collagen triple helix depends on the identity and sequence of the amino acid residues Xaa and Yaa in the Gly-Xaa-Yaa sequence. H-Bonding on the interior of the triple helix holds the triple helix together. The imino acids, proline (Pro) and hydroxyproline (Hyp), are both stabilizing factors, so that the melting temperature Tm of collagen from many animals is proportional to the imino acid content (2). In general, the higher the Hyp content in the Yaa position, the higher the Tm. In addition, it was shown that amino acids such as 4-fluoroproline or 4-aminoproline influence the Tm, causing it to increase due to electronic effects (3). This may have interesting application for the formation of biomaterials that exhibit a higher stability under biological conditions (4). On the basis of the biophysical studies on (Gly-Pro-Pro)10, Bhatnagar and co-workers suggested that the major stabilizing force in the Gly-Pro-Pro triple-helix is the interchain and/or interresidue nonbonded interactions, including close van der Waals contacts and hydrophobic interactions (5). Goodman and co-workers demonstrated that template-assembled collagen residues greatly enhance the conformational stability of the collagen triple helix, and recently they also reported the incorporation of an Fe3+-catechol complex as a scaffold to assemble peptide chains into triple helices (6). As a part of our ongoing study into the electron transfer properties of peptides chemically linked to gold surfaces, we synthesized a series of Fc-labeled collagen mimetic structures composed of Pro-HypGly sequences (Scheme 1) equipped with a C-terminal Cys residue and began to assess the propensity for triple helix formation by variable temperature circular dichroism (CD) spectroscopy, thermodynamic studies, and molecular modeling. Here we present the results of this study indicating that Fc, a simple organometallic molecule, attached to the N-terminal end of the collagen helix enhances the stability of the triple helix. * To whom correspondence should be addressed. Fax: 306-9664730; Tel: 306-966-4660. E-mail: [email protected].

MATERIALS AND METHODS All chiral amino acids were of the L configuration. Amino acids, SPPS resins, and HOBt were purchased from SynPep. EDC was purchased from Novabiochem. DIEAP, TFA (HPLCgrade), TIS, piperidine, phenol, and ferrocene were purchased from Aldrich Chem. DMF (BDH; ACS grade) was used for synthesis. Acetone, CH2Cl2, diethyl ether (BDH; ACS grade), CH3CN (Fischer; HPLC grade), and CH3OH (Fisher, for electrochemistry, spectra-grade) were used for the purpose of purification. Reagents were used as provided. All solutions (for biophysical studies and HPLC purification) were prepared using deionized Millipore water (18.2 MΩ‚cm) obtained from a Milli-Q water system. General Procedure. A series of collagen models (both the modified model with ferrocene and unmodified model) were synthesized by the standard N-(9-fluorenyl)methoxycarbonyl (Fmoc)-based solid-phase protocol on a Quest Organic Synthesizer. Couplings were carried out on Fmoc-L-cysteine(trityl) Wang resin (Syn Pep, 0.50 mmol/g) using Fmoc amino acids (Fmoc-Gly-OH, Fmoc-Hyp-OH, and Fmoc-Pro-OH, 2 equiv each) in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) (2.2 equiv) and N-hydroxybenzotriazole (HOBt) (2.2 equiv) as activating agents and diisopropylethylamine (4 equiv). To prepare ferrocene-attached collagen-like polypeptides, the specific peptide chains were built up on resin, and the ferrocene monocarboxylic acid was directly coupled to the N-termini of these chains before removal from the resin. The synthetic route is described in Scheme 2. Coupling efficiency was monitored with Kaiser test (7), except for N-terminal proline residues where the chloranil test (8) was applied. The desired peptides were cleaved from the resin with trifluoroacetic acid (TFA) containing phenol (2.5% v/v) and triethylsilane (TIS; 2.5% v/v) and then precipitated with diethyl ether.

10.1021/bc050268l CCC: $33.50 © 2006 American Chemical Society Published on Web 12/31/2005

Ferrocene-Labeled Collagen Mimetic

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Scheme 2

Peptides are purified by reversed-phase high performance liquid chromatography (RP-HPLC) (Varian, Inc.) using a water-acetonitrile system and Vydac C-18, 25 × 2.2 cm preparatory column. Peptides were eluted with a gradient of aqueous acetonitrile containing TFA (0.1%, v/v) at a flow rate of 16.6 mL/min. Purified peptides were obtained by collecting the major peak with absorbance at 215 nm, which occurred between 32 and 77% acetonitrile. HPLC-purified peptides were analyzed by electrospray mass spectrometry using a QSTAR XL-MS/MS ionspray source. Synthesis and Purification. The Fmoc-protected Wang-Cys resin (loaded: 0.50 mmol/g, taken 0.1 mmol) was treated with 25% piperidine in DMF (1 × 20 min, 1 × 10 min) and then washed successively with DMF (3 × 1 min), MeOH (3 × 1 min), and CH2Cl2 (3 × 1 min). Coupling of the first amino acid (Fmoc-Gly-OH) was performed with Fmoc-Gly-OH/EDC/ HOBt/DIEA (2 equiv, 1:1.1:1.1:2) in DMF (2.5h) followed by washing with DMF (3 × 1 min), MeOH (3 × 1 min), and CH2Cl2 (3 × 1 min). Chain elongation was performed with FmocHyp-OH/EDC/HOBt/DIEA (2 equiv, 1:1.1:1.1:2) and FmocPro-OH/EDC/HOBt/DIEA (2 equiv, 1:1.1:1.1:2) sequentially up to the desired length. Fmoc cleavage from Fmoc-Pro-Peptidyl resin was carried out with 25% piperidine and 1% DBU in DMF (2 × 15 min) and washings with DMF (3 × 1 min), MeOH (3 × 1 min), and CH2Cl2 (3 × 1 min). In the final step, half of the polypeptide resin beads were taken out, the desired polypeptides were cleaved from the resin with trifluoroacetic acid (TFA) containing phenol (2.5% v/v) and triisopropylsilane (TIS; 2.5% v/v), and with the remainder of the beads, an additional Fccoupling step was carried out. Fc-COOH/EDC/HOBt/DIEA (2 equiv, 1:1.1:1.1:2) was added to the remaining beads. The Fclabeled collagen polypeptides were also cleaved from the resin using the same cleaving agent. All modified and unmodified collagen models were purified by RP-HPLC using a wateracetonitrile gradient and characterized by ESI-MS spectra. For the spectroscopic and electrochemical measurements, a portion of the material was further purified by preparative HPLC (ESI). CD Spectroscopy. Circular dichroism (CD) spectra were recorded on a π*-180 spectrometer (Applied Photophysics Ltd, UK) equipped with a model 3016 isotemp refrigerator circulator (Fisher Scientific), interfaced to an Acorn PC. A rectangular 2

mm-path length cell was used. The spectrometer was routinely calibrated with an aqueous solution of (1S)-(+)-10-camphorsulfonic acid. Mean residue molar ellipticities were calculated according to the following equation:

[θ] ) [θ]obs/10 × lcn where [θ]obs is the observed ellipticity measured in degrees, l is the path length of the cell in centimeters, c is the molar peptide concentration, and n is the number of amino acid residues in the peptide. The spectra for both modified and unmodified polypeptides were obtained as an average of eight scans using a slit width of 0.5 nm. The thermal denaturation results were obtained by measuring the ellipticity at 223 nm as a function of temperature using a 120 s thermal equilibration time between data points.

RESULTS AND DISCUSSION In addition to nonnatural amino acids, one can expect that increasing the hydrophobicity at the N-terminal side of the collagens may also influence the melting behavior. In our efforts toward the design of collagen conjugates, we have synthesized a series of Fc-labeled collagen mimetic structures composed of Pro-Hyp-Gly sequences (Scheme 1) to assess their triple helical propensities and the role of Fc in triple helix stability by variable temperature circular dichroism (CD) spectroscopy, thermodynamic studies, and molecular modeling. Fc-collagen conjugates Fc-CO-(Pro-Hyp-Gly)n-Cys (n ) 4 (1), 6 (2), 7 (3), 8 (4), 9 (5)) of increasing peptide length were synthesized by solid-phase synthesis on Wang resin using EDC/ HOBt, cleaved off the resin by TFA, phenol, and triethylsilane (95:2.5:2.5), and then purified by preparative RP-HPLC on C18. All compounds were characterized by mass spectroscopy. The synthesis, purification, and characterization of the unlabeled collagens (lacking the Fc group) were carried out in the same fashion (Scheme 2). In water/methanol (99/1), compounds 1-5 exhibit a single broad absorbance in the visible region with a λmax between 430 and 445 nm. For all of the complexes excitation within the region of 200-240 nm, the peptide bond

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Figure 1. Modeling of the Fc-labeled collagen mimics (for compound 5).

due to the n f π * transitions yields UV spectra dominated by the peptide bond absorptions. The solution electrochemistry in water/methanol (99/1) for compounds 1-5 shows a fully reversible oxidation wave of the Fc group with a half-wave potential E1/2 at about 560 mV, a peak separation of 70 mV, and a ratio of anodic to cathodic peak currents close to unity (ESI, Table S1). Molecular Modeling. Molecular models of triple-helical ferrocene-labeled compounds were created by modifying the structure of crystalline collagen (PDB entry 1CGD (9)). The side chain methyl group of the three alanine residues was replaced by a hydrogen atom. The one (Pro-Hyp-Gly) unit was deleted from the C-terminus (for 5, Figure 1). This section was subjected to energy minimization for equilibrium geometry using Hartree-Fock method. Three Fc-CO groups were added to the N-terminal proline residues, and three Cys groups were added to the C-terminal glycine residues. The whole section was subjected to energy minimization, locking all the entire amide bonds by Hartree-Fock method except the cysteine amide group. The molecular modeling of ferrocene-labeled triple helices includes two basic steps. First, the backbones of three Fc-CO[Pro-Hyp-Gly]n-Cys (where n ) 4, 6-9) chains have to be assembled into a triple-helical assembly. Second, the conformational space accessible to the ferrocene group needs to be searched for minimum energy structures. The first step concerns only the chain backbone and simplifies the process of triplehelical assembly. The three Fc-CO-[Pro-Hyp-Gly]n-Cys molecules were constrained to have the same backbone torsions as the structure proposed by Scheraga (10) for (Gly-Pro-Hyp)n. This conformation has been shown to be very stable with respect to the unsubstituted compounds and is therefore possibly a good candidate to use as starting backbone conformation for minimizations. On the basis of our present study, we conclude that the Fc-collagen conjugates have an enhanced stability toward denaturation.

Determination of Triple-Helical Conformation in Solution. The solution conformation of compounds 1-5 was investigated by CD spectroscopy. Natural collagen has a CD spectrum exhibiting a low intensity maximum at 220 nm, a crossover at 213 nm, and a minimum at 197 nm (11). Using these data, the helicity of any collagen can be determined. The CD spectra for compounds 1-5 are shown in Figure 2. All compounds display a spectrum typical of collagen with a maximum at 223, a minimum at 201 nm, and a crossover at 218 nm. The spectral data is listed in Table 1 and is comparable to known collagen models, (Gly-Pro-Hyp)10-NH2 (11, 12). As the length of the peptide increases in compounds 1-5, the helical content increases up to the reported value for (Gly-Pro-Hyp)10-OH (12c). The entire unmodified system also demonstrates a similar type

Figure 2. CD spectra of Fc-CO-(Pro-Hyp-Gly)n-Cys where n ) 4, 6-9 in water/methanol, 99/1, at pH 7.2 at 20 °C. Blue line for n ) 9, green for n ) 8, black for n ) 7, red for n ) 6, and orange for n ) 4. The samples were kept in a refrigerator (4 °C) for at least 1 week so they would reach the equilibrium of triple-helix formation.

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Table 1. Circular Dichroism Data for the Synthesized Fc-attached Collagen-Based Polypeptides Composed of (Pro-Hyp-Gly) Sequencesa compound

λmin (nm)

cross (nm)

λmax (nm)

Rpn

1 2 3 4 5 collagenb (G-P-O)10-OH c

202 (-1.96 × 104) 201 (-4.06 × 104) 200 (-4.06 × 104) 201 (-4.11 × 104) 201 (-4.93 × 104) 198 (-5.4 × 104) 198 (-3.4 × 104)

219 218 216 217 216 213 218

223 (0.22 × 104) 223 (0.37 × 104) 223 (0.63 × 104) 223 (0.63 × 104) 223 (0.87 × 104) 220 (0.71 × 104) 225 (0.43 × 104)

0.11 0.09 0.14 0.14 0.17 0.13 0.13

b,c

Tm (°C)

Tm (°C, unmodified)

∆Hm, kJ mol-1

∆Sm, kJ mol-1 K-1

length (Å)

38 ( 3 48 ( 1 50 ( 2 70 ( 1 73 ( 1

31 ( 1 38 ( 2 39 ( 1 51 ( 3 66 ( 2

10 34 72 103 154

0.03 0.10 0.22 0.30 0.45

32 48 56 64 72

a CD spectra were obtained at 20 °C using a peptide concentration of 0.01 mM (water/methanol, 99/1). The peak intensities are indicated in the parentheses. Estimated from published CD spectra: ref 12b,c.

Figure 3. Thermal denaturation of ferrocene-labeled collagen triple helices at 223 nm (blue line for n ) 9, green for n ) 8, black for n ) 7, red for n ) 6, and orange for n ) 4). The critical triple-helical concentration is ca. 0.01 mM (methanol/water). Ellipticities at 223 nm were monitored by circular dichroism spectroscopy as the temperature was increased by increments of 5 °C with 2-min equilibrium.

of spectrum with a maximum at 222 nm and a minimum at around 200 nm (e.g., compound 5, modified and unmodified systems, ESI, Figure S1). To establish the triplex formation we have used the ratio of the positive peak intensity over negative peak intensity, Rpn (12c, 13). For the synthetic Fc-collagen conjugates 1-5, the Rpn values are summarized in Table 1, including the values for the natural collagen (13) and for (GlyPro-Hyp)10-OH (11c). On the basis of these Rpn values, the presence of triple-helical conformations is established for our ferrocene-labeled systems 1-5 at 20 °C and a concentration of 0.01 mM at pH 7.2. As the solution temperature was gradually increased from 5 to 100 °C, the CD spectrum for compounds 1-5 change gradually, showed a pattern featuring the triplehelix polypeptide with slight damage to the triple helix at higher temperatures. The positive Cotton effect disappeared at 223 nm (ESI, Fig. S2), which reflects the loosening of the triple helix and dissociation to single helical strands having polyproline-II conformations. The resulting Tm value provides an indication of the conformational stability of a triple helix (Figure 3). The key result of our study is that the temperature of the triplex-to-singlex transition is higher for the Fc-collagen conjugates 1-5 than for the collagens lacking the N-terminal Fc group, indicating that the Fc group is stabilizing the triplex. The resulting Tm values, along with those of triple helices of same number of amino acids (lengths (in Å) for all of complexes were calculated from molecular modeling) without the Fc moiety are tabularized in Table 1 and compared in Figure 4 and Figure 5. This indicates that the modification of collagen by the Fc group stabilizes the triple helix, which can be due to changes in the local chemical environment, such as the additional hydrogen bond and/or hydrophobic interaction or the overall conformational change

Figure 4. Thermal denaturation of ferrocene-labeled collagen triple helices at 223 nm ((g) for modified system, Compd. 5, and (O) for unmodified system of same length). The critical triple-helical concentration is ca. 0.01 mM (methanol/water). Ellipticities at 223 nm were monitored by circular dichroism spectroscopy as the temperature was increased by increments of 5 °C with 2-min equilibrium in both cases.

Figure 5. Comparison of thermal denaturation values of ferrocenelabeled collagen triple helices with unlabeled systems at 223 nm (star for the Fc-labeled systems, block for unlabeled systems).

including the whole Pro-Hyp-Gly moiety, which is induced by attachment of the hydrophobic Fc group. Importantly, we observe a small hysteresis of the melting behavior, shown in Figure 6. Although, the overall features in the CD spectra recovered after heating and cooling of aqueous solutions of compounds 1-5, the intensities of the bands were diminished. However, the positive and negative Cotton effects did not completely recover their intensities, but it proves that the conformational change in the triple helices was nearly reversible in aqueous solution.

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at different temperatures, table for thermodynamic data, details of thermal unfolding, data analysis (e.g., compound 5). This material is available free of charge via the Internet at http:// pubs.acs.org.

LITERATURE CITED

Figure 6. Thermal unfolding (g) and refolding (0) of ferrocenelabeled collagen model (5) at 0.01 mM concentration in aqueous solution (pH 7.2) as monitored by CD at 223 nm. Melting and formation of the triple helix are virtually reversible.

Thermodynamic parameters are also estimated from CD transition profiles at 223 nm to understand the overall nature of the thermal stability of our Fc-labeled systems (ESI, Figures S3, S4). In thermal unfolding experiments, the thermodynamic parameters of the compounds 1-5 were calculated assuming two-state denaturation process (14) using Levenberg-Marquardt nonlinear least-squares method. The van’t Hoff enthalpy, ∆Hm, was calculated by nonlinear least-squares fitting of the CD transition profile. The entropy change of the transition at Tm, ∆Sm, was calculated by dividing ∆Hm by Tm (see Table 1). Expectedly, ∆Hm increases with increasing the collagen chain length, which indicates an increase in stability as the collagen chain length increases. It is noteworthy to point out that this trend is reflected also in ∆Hm for Fc-labeled and unlabeled collagens (see ESI, Table S2, for n ) 9), which again emphasizes that Fc-collagens appear stabilized compared to the corresponding unlabeled collagens in different ways. However, it should be noted that errors higher than 10% seem to be unavoidable for parameters derived from the van’t Hoff equation.

CONCLUSION We have presented the results of our comparative study showing that the attachment of an Fc group to the N-terminus of a collagen peptide stabilizes the formation of the collagen triple helix. These findings open an unexpected path for the design of stable and redox active collagen mimics and in the use of collageneous peptides for the studies on collagen structure, folding, and biochemistry. We are currently synthesizing some Fc-labeled collagen models in which individual amino acids are replaced. We are also investigating the possibility of electron-transfer reactions in Fc-labeled collagen films containing redox entities with a cysteine linker.

ACKNOWLEDGMENT The Natural Science and Engineering Research Council of Canada supported this research. H.-B.K. is the Canada Research Chair in Biomaterials. Our thanks are also extended to Mr. Somenath Choudhury, Dr. Yitao Long, and Dr. Todd Sutherland Department of Chemistry, University of Saskatchewan, for their valuable discussion in the circular dichroism studies. Supporting Information Available: HPLC traces and ESI-MS, solution CVs of Fc-labeled collagens and the CD spectrum of a modified and unmodified collagen system, the CD spectrum

(1) Baum, J., and Brodsky, B. (2000) In Mechanisms of Protein Folding, 2nd ed. (Pain, R. H., Ed.) pp 330-351, Oxford University Press, Oxford. (2) (a) Rich, A., and Crick, F. H. C. (1961) The Molecular Structure of Collagen. J. Mol. Biol. 3, 483-506. (b) Ramachandran, G. N. (1964) In Treatise on Collagen (Ramachandran, G. N., Ed.) pp 103183, Academic Press: New York. (c) Radmer, R. J., and Klein, T. E. (2004) Severity of Osteogenesis Imperfecta and Structure of a Collagen-like Peptide Modeling a Lethal Mutation Site. Biochemistry 43, 5314-5323. (d) Persikov, A. V., Ramshaww, J. A. M., Kirkpatrick, A., and Brodsky, B. (2003) Triple-Helix Propensity of Hydroxyproline and Fluoroproline: Comparison of Host-Guest and Repeating Tripeptide Collagen Models. J. Am. Chem. Soc. 125, 11500-11501. (e) Hodges, J. A., and Ranies, R. T. (2003) Stereoelectronic Effects on Collagen Stability: The Dichotomy of 4-Fluoroproline Diastereomers. J. Am. Chem. Soc. 125, 9262-9263. (f) Doi, M., Nishi, Y., Uchiyama, S., Nakazawa, T., Ohkubo, T., and Kobayashi, Y. (2003) Characterization of Collagen Model Peptides Containing 4-Fluoroproline; (4(S)-Fluoroproline-Pro-Gly)10 Forms a Triple Helix, but (4(R)-Fluoroproline-Pro-Gly)10 Does Not. J. Am. Chem. Soc. 125, 9922-9923. (g) Berisio, R., Granata, V., Vitagliano, L., and Zagari, A. (2004) Imino Acids and Collagen Triple Helix Stability: Characterization of Collagen-like Polypeptides Containing Hyp-Hyp-Gly Sequence Repeats. J. Am. Chem. Soc. 126, 11402-11403. (3) (a) Holmgren, S. K., Taylor, K. M., Brestscher, L. E., and Ranies, R. T. (1998) Code for collagen’s stability deciphered. Nature 392, 666-667. (b) Umashankara, M., Babu, I. R., and Ganesh, K. N. (2003) Two prolines with a difference: contrasting stereoelectronic effects of 4R/S-aminoproline on triplex stability in collagen peptides [Pro(X)-Pro(Y)-Gly]n. Chem. Commun. 2606-2607. (4) Koide, T., Yuguchi, M., Kawakita, M., and Konno, H. (2002) Metal-Assisted Stabilization and Probing of Collagenous Triple Helices. J. Am. Chem. Soc. 124, 9388-9399. (5) Bhatnagar, R. S., and Gough, C. A. (1996) in Circular Dichroism and the Conformational Analysis of Biomolecules (Fasman, G. D., Ed.) pp 183-199, Plenum Press, New York. (6) (a) Feng, Y., Melacini, G., Taulane, J. P., and Goodman, M. (1996) Acetyl-Terminated and Template-Assembled Collagen-Based Polypeptides Composed of Gly-Pro-Hyp Sequences. 2. Synthesis and Conformational Analysis by Circular Dichroism, Ultraviolet Absorbance, and Optical Rotation. J. Am. Chem. Soc. 118, 10351-10358. (b) Feng, Y., Melacini, G., and Goodman, M. (1997) Collagen-Based Structures Containing the Peptoid Residue N-Isobutylglycine (Nleu): Synthesis and Biophysical Studies of Gly-Nleu-Pro Sequences by Circular Dichroism and Optical Rotation. Biochemistry 36, 8716-8724. (c) Cai, W., Kwok, S. W., Taulane, J. P., and Goodman, M. (2004) Metal-assisted Assembly and Stabilization of Collagen-like Triple Helices. J. Am. Chem. Soc. 126, 15030-15031. (7) Kaiser, E., Colescott, R. L., Bossinger, C. D., and Cook, P. I. (1970) Color test for detection of free terminal amino groups in the solidphase synthesis of peptides. Anal. Biochem. 34, 595-598. (8) Christensen, T. (1979) Peptides, Structure and Biological Functions. Proceedings of the 6th American Peptide Symposium (Gross, E., Meienhofer, J., Eds.) pp 385-388, Pierce Chemical Co., Rockford, IL. (9) Bella, J., Brodsky, B., and Bermanm, H. M. (1995) Hydration Structure of a Collagen Peptide. Structure 3, 893-906. (10) Miller, M. H., Nemethy, G., and Scheraga, G. H. A. (1980) Calculations of the Structures of Collagen Models. Role of Interchain Interactions in Determining the Triple-Helical Coiled-Coil Conformation. 2. Poly(glycyl-prolyl-hydroxyprolyl). Macromolecules 13, 470-478. (11) (a) Sakakibara, S., Kishida, Y., Okuyama, K., Tanaka, N., Ashida, T., and Kakudo, M. (1972) Single crystals of (Pro-Pro-Gly)10, a synthetic polypeptide model of collagen. J. Mol. Biol. 65, 371-

Bioconjugate Chem., Vol. 17, No. 1, 2006 89

Ferrocene-Labeled Collagen Mimetic 372. (b) Brown, F. R., III, Corato, A. Di, Lorenzi, G. P., and Blout, E. R. (1972) Synthesis and structural studies of two collagen analogues: Poly (L-prolyl-L-seryl-glycyl) and poly (L-prolyl-Lalanyl-glycyl). J. Mol. Biol. 63, 85-99. (c) Toumadje, A., and Johnson, W. C., Jr. (1995) Systemin has the characteristics of a poly(L-proline) II type helix. J. Am. Chem. Soc. 117, 7023-7024. (12) (a) Jenkins, C. L., and Ranies, R. T. (2002) Insights on the conformational stability of collagen. Nat. Prod. Rep. 19, 49-59. (b) Periskov, A. V., Ramshaw, J. A. M., and Brodsky, B. (2000) Collagen model peptides: Sequence dependence of triple-helix stability. Pept. Sci. 55, 436-450. (c) Venugopal, M. G., Ramshaw, J. A. M., Braswell, E., Zhu, D., and Brodsky, B. (1994) Electrostatic

Interactions in Collagen-like Triple-Helical Peptides. Biochemistry 33, 7948-7956. (13) (a) Brown, F. R., III, Carver, J. P., and Blout, E. R. (1969) Low temperature circular dichroism of poly (glycyl-L-prolyl-L-alanine). J. Mol. Biol. 39, 307-313. (b) Brown, F. R., III, Hopfinger, A. J., and Blout, E. R. (1972) The collagen-like triple helix to randomchain transition: Experiment and theory. J. Mol. Biol. 63, 101115. (14) Kizhakkedathu, A. K. J. P., Nazeerunnisa, M., and Behere, D. V. (2002) Thermal Unfolding of Soybean Peroxidase. J. Biol. Chem. 277, 40717-40721. BC050268L