ABSTRACT Here we present the cloning, expression and immunocytochemical localization of a novel 24 kDa protein, designated spinalin, which is present in the spines and operculum of Hydra nematocysts. Spinalin cDNA clones were identified by in situ hybridization to differentiating nematocytes. Sequencing of a full-length clone revealed the presence of an N-terminal signal peptide, suggesting that the mature protein is sorted via the endoplasmic reticulum to the post-Golgi vacuole in which the nematocyst is formed. The N-terminal region of spinalin (154 residues) is very rich in glycines (48 residues) and histidines (33 residues). A central region of 35 residues contains 19 glycines, occurring mainly as pairs. For both regions a polyglycine-like structure is likely and this may be stabilized by hydrogen bond-mediated chain association. Similar sequences found in loricrins, cytokeratins and avian keratins are postulated to participate in formation of supramolecular structures. Spinalin is terminated by a basic region (6 lysines out of 15 residues) and an acidic region (9 glutamates and 9 aspartates out of 32 residues). Western blot analysis with a polyclonal antibody generated against a recombinant 19 kDa fragment of spinalin showed that spinalin is localized in nematocysts. Following dissociation of the nematocyst’s capsule wall with DTT, spinalin was found in the insoluble fraction containing spines and the operculum. Immunocytochemical analysis of developing nematocysts revealed that spinalin first appears in the matrix but then is transferred through the capsule wall at the end of morphogenesis to form spines on the external surface of the inverted tubule and the operculum.
Collagen consists of repetitive Gly-Xaa-Yaa tripeptide units with proline and hydroxyproline frequently found in the Xaa and Yaa position, respectively. This sequence motif allows the formation of a highly regular triple helix that is stabilized by steric (entropic) restrictions in the constituent polyproline-II-helices and backbone hydrogen bonds between the three strands. Concentration-dependent association reactions and slow prolyl isomerization steps have been identified as major rate-limiting processes during collagen folding. To gain information on the dynamics of triple-helix formation in the absence of these slow reactions, we performed stopped-flow double-jump experiments on cross-linked fragments derived from human type III collagen. This technique allowed us to measure concentration-independent folding kinetics starting from unfolded chains with all peptide bonds in the trans conformation. The results show that triple-helix formation occurs with a rate constant of 113 +/- 20 s(-1) at 3.7 degrees C and is virtually independent of temperature, indicating a purely entropic barrier. Comparison of the effect of guanidinium chloride on folding kinetics and stability reveals that the rate-limiting step is represented by bringing 10 consecutive tripeptide units (3.3 per strand) into a triple-helical conformation. The following addition of tripeptide units occurs on a much faster time scale and cannot be observed experimentally. These results support an entropy-controlled zipper-like nucleation/growth mechanism for collagen triple-helix formation.
Abstract Specific cell–cell contacts are important in tissue morphogenesis, development, and during signal transduction of vertebrates as well as invertebrates. Cadherins are cell‐surface transmembrane receptors that mediate such specific, homotypic cell–cell adhesion. The adhesive activity of cadherins requires calcium binding by the ectodomain of the protein. The extracellular part of cadherins is composed of different numbers of individually folding cadherin repeats (CAD). Calcium binding pockets are located in the interdomain sections and conserved peptide sequences in consecutive domains are involved in coordination of three calcium ions per binding pocket. Low‐ and high‐affinity calcium binding sites have been detected and binding constants in the micromolar ( K d = 30–330 µM) to millimolar ( K d = 2 mM) range were determined. When calcium is added to purified cadherin ectodomains, rigidification and conformational changes are observed, exposing interaction surfaces that are involved in homoassociation of cadherins. This review summarizes electron micrographic, crystallographic, and recent NMR data analyzing the molecular details and functional consequences of calcium binding by cadherins.
Type III collagen is a critical collagen that comprises extensible connective tissue such as skin, lung, and the vascular system. Mutations in the type III collagen gene, COL3A1, are associated with the most severe forms of Ehlers-Danlos syndrome. A characteristic feature of type III collagen is the presence of a stabilizing C-terminal cystine knot. Crystal structures of collagen triple helices reported so far contain artificial sequences like (Gly-Pro-Pro)n or (Gly-Pro-Hyp)n. To gain insight into the structural properties exhibited by the natural type III collagen triple helix, we synthesized, crystallized, and determined the structure of a 12-triplet repeating peptide containing the natural type III collagen sequence from residues 991 to 1032 including the C-terminal cystine knot region, to 2.3Å resolution. This represents the longest collagen triple helical structure determined to date with a native sequence. Strikingly, the Gly991–Gly1032 structure reveals that the central non-imino acid-containing region adopts 10/3 superhelical properties, whereas the imino acid rich N- and C-terminal regions adhere to a 7/2 superhelical conformation. The structure is consistent with two models for the cystine knot; however, the poor density for the majority of this region suggests that multiple conformations may be adopted. The structure shows that the multiple non-imino acids make several types of direct intrahelical as well as interhelical contacts. The looser superhelical structure of the non-imino acid region of collagen triple helices combined with the extra contacts afforded by ionic and polar residues likely play a role in fibrillar assembly and interactions with other extracellular components. Type III collagen is a critical collagen that comprises extensible connective tissue such as skin, lung, and the vascular system. Mutations in the type III collagen gene, COL3A1, are associated with the most severe forms of Ehlers-Danlos syndrome. A characteristic feature of type III collagen is the presence of a stabilizing C-terminal cystine knot. Crystal structures of collagen triple helices reported so far contain artificial sequences like (Gly-Pro-Pro)n or (Gly-Pro-Hyp)n. To gain insight into the structural properties exhibited by the natural type III collagen triple helix, we synthesized, crystallized, and determined the structure of a 12-triplet repeating peptide containing the natural type III collagen sequence from residues 991 to 1032 including the C-terminal cystine knot region, to 2.3Å resolution. This represents the longest collagen triple helical structure determined to date with a native sequence. Strikingly, the Gly991–Gly1032 structure reveals that the central non-imino acid-containing region adopts 10/3 superhelical properties, whereas the imino acid rich N- and C-terminal regions adhere to a 7/2 superhelical conformation. The structure is consistent with two models for the cystine knot; however, the poor density for the majority of this region suggests that multiple conformations may be adopted. The structure shows that the multiple non-imino acids make several types of direct intrahelical as well as interhelical contacts. The looser superhelical structure of the non-imino acid region of collagen triple helices combined with the extra contacts afforded by ionic and polar residues likely play a role in fibrillar assembly and interactions with other extracellular components. Collagens are the most abundant proteins in animals, comprising an estimated one-third of the total protein by weight (1Myllyharju J. Matrix Biol. 2003; 22: 15-24Crossref PubMed Scopus (338) Google Scholar, 2Myllyharju J. Kivirikko K.I. Trends Genet. 2004; 20: 33-43Abstract Full Text Full Text PDF PubMed Scopus (894) Google Scholar, 3Kielty M.E. Royce P.M. Steinmann B. Connective Tissue and Its Heritable Disorders. 2nd Ed. Wiley-Liss, New York1993Google Scholar). At least 27 collagen types, which are formed from 42 distinct polypeptide chains, exist in vertebrates (1Myllyharju J. Matrix Biol. 2003; 22: 15-24Crossref PubMed Scopus (338) Google Scholar, 2Myllyharju J. Kivirikko K.I. Trends Genet. 2004; 20: 33-43Abstract Full Text Full Text PDF PubMed Scopus (894) Google Scholar). In addition, more than 20 additional proteins adopt collagen-like structures such as collectins, ficolins, and scavenger receptors (2Myllyharju J. Kivirikko K.I. Trends Genet. 2004; 20: 33-43Abstract Full Text Full Text PDF PubMed Scopus (894) Google Scholar). Collagen is an essential molecule in vertebrates, because it plays the dominant role in maintaining the structure of tissues. However, collagen and collagen-like proteins have many other important roles, such as cell adhesion, chemotaxis, cell migration, and the regulation of tissue remodeling during cell growth, differentiation, morphogenesis, and wound healing (2Myllyharju J. Kivirikko K.I. Trends Genet. 2004; 20: 33-43Abstract Full Text Full Text PDF PubMed Scopus (894) Google Scholar). All collagen molecules consist of three polypeptide chains, called α chains, which contain at least one region of repeating Gly-Xaa-Yaa sequences (1Myllyharju J. Matrix Biol. 2003; 22: 15-24Crossref PubMed Scopus (338) Google Scholar, 2Myllyharju J. Kivirikko K.I. Trends Genet. 2004; 20: 33-43Abstract Full Text Full Text PDF PubMed Scopus (894) Google Scholar). In the collagen molecule, the three α chains each fold into a polyproline II-like left-handed structure, and the three polyproline II-like chains twist around each other to form a right-handed superhelix, called the collagen triple helix (4Rich A. Crick F.H. Nature. 1955; 176: 915-916Crossref PubMed Scopus (319) Google Scholar, 5Brodsky B. Persikov A.V. Adv. Protein Chem. 2005; 70: 301-339Crossref PubMed Scopus (443) Google Scholar, 6Okuyama K. Wu G. Jiravanichanun N. Hongo C. Noguchi K. Biopolymers. 2006; 84: 421-432Crossref PubMed Scopus (49) Google Scholar, 7Okuyama K. Xu X. Iguchi M. Noguchi K. Biopolymers. 2006; 84: 181-191Crossref PubMed Scopus (75) Google Scholar). Critical to the formation of the triple helix is the presence of a glycine residue at each third position in the chain because this residue is the only one that can exist in the small space at the center of the triple helix (8Prockop D.J. Kivirikko K.I. Tuderman L. Guzman N.A. N. Engl. J. Med. 1979; 301: 13-23Crossref PubMed Scopus (750) Google Scholar). Each of the three chains therefore has the repeating structure Gly-Xaa-Yaa, in which Xaa and Yaa can be any amino acid but are frequently the imino acids proline in the Xaa position and hydroxyproline (Hyp) in the Yaa position. Because both proline and Hyp are rigid, cyclic imino acids, they limit rotation of the polypeptide backbone and thus contribute to the stability of the triple helix. Collagen polypeptides that lack Hyp can fold into a triple helical conformation at low temperatures, but the triple helix formed is not stable at mammalian body temperature (8Prockop D.J. Kivirikko K.I. Tuderman L. Guzman N.A. N. Engl. J. Med. 1979; 301: 13-23Crossref PubMed Scopus (750) Google Scholar). The number of Gly-Pro-Hyp repeats is the main, but not exclusive, factor in determining collagen thermostability (9Burjanadze T.V. Biopolymers. 2000; 53: 523-528Crossref PubMed Scopus (85) Google Scholar). Approximately 90% of collagen tripeptide units contain at least one non-imino acid residue in the Xaa and/or Yaa position, and these residues likely play a role in collagen structure, stability, and function (5Brodsky B. Persikov A.V. Adv. Protein Chem. 2005; 70: 301-339Crossref PubMed Scopus (443) Google Scholar, 6Okuyama K. Wu G. Jiravanichanun N. Hongo C. Noguchi K. Biopolymers. 2006; 84: 421-432Crossref PubMed Scopus (49) Google Scholar, 7Okuyama K. Xu X. Iguchi M. Noguchi K. Biopolymers. 2006; 84: 181-191Crossref PubMed Scopus (75) Google Scholar). Indeed, a notable feature of the collagen triple helix is that the amino acids occupying Xaa and Yaa positions are solvent-accessible. Because of this, these residues would be predicted to play important roles in interactions with other molecules, such as extracellular matrix proteins. In addition, these residues are predicted to be important in collagen triple helix self-association leading to fibril formation. The best characterized and most common collagen fibril form is the 67-nm (D) periodic fibril, which is observed in most connective tissues (10Bruns R.R. Gross J. Biochemistry. 1973; 12: 808-815Crossref PubMed Scopus (81) Google Scholar, 11Doyle B.B. Hulmes D.J.S. Miller A. Parry D.A.D. Piez K.A. Woodhead-Galloway J. Proc. R. Soc. Lond. Ser. B Biol. Sci. 1974; 187: 37-46Crossref PubMed Scopus (55) Google Scholar). Collagen types I, II, III, V, and XI self-associate to form these characteristic fibrils. Studies suggest that the axial staggering of collagen molecules that give rise to these fibrils is due to electrostatic and hydrophobic interactions between neighboring molecules (12Hulmes D.J.S. Miller A. Parry D.A.D. Piez K.A. Woodhead-Galloway J. J. Mol. Biol. 1973; 79: 137-148Crossref PubMed Scopus (344) Google Scholar, 13Li S.-T. Golub E. Katz E.P. J. Mol. Biol. 1975; 98: 835-839Crossref PubMed Scopus (27) Google Scholar, 14Trus B.L. Piez K.A. J. Mol. Biol. 1976; 108: 705-732Crossref PubMed Scopus (41) Google Scholar). Specifically, oppositely charged residues in the Xaa and Yaa position of the collagen triple helix are predicted to play a role in determining the axial stagger of the fibril (11Doyle B.B. Hulmes D.J.S. Miller A. Parry D.A.D. Piez K.A. Woodhead-Galloway J. Proc. R. Soc. Lond. Ser. B Biol. Sci. 1974; 187: 37-46Crossref PubMed Scopus (55) Google Scholar). Such charged residues have also been implicated in the interactions between macrophage scavenger receptor and other molecules and the hexamer formation of the serum complement protein C1q (15Acton S. Resnick D. Freeman M. Ekkel Y. Ashkenas J. Krieger M. J. Biol. Chem. 1993; 268: 3530-3537Abstract Full Text PDF PubMed Google Scholar, 16Doi T. Higashino K.-I. Kurihara Y. Wada Y. Miyazaki T. Nakamura H. Uesugi S. Imanishi T. Kawabe Y. Itakura H. J. Biol. Chem. 1993; 268: 2126-2133Abstract Full Text PDF PubMed Google Scholar, 17Hoppe H.-J. Reid K.B.M. Protein Sci. 1994; 3: 1143-1158Crossref PubMed Scopus (191) Google Scholar). Type III collagen contains multiple charged residues in the Xaa and Yaa positions of its chain (18Ala-Kokko L. Kontusaari S. Baldwin C.T. Kuivaniemi H. Prockop D.J. Biochem. J. 1989; 260: 509-516Crossref PubMed Scopus (116) Google Scholar). Type III collagen is the second most abundant collagen in human tissues after type I and is primarily found in tissues exhibiting elastic properties, such as skin, blood vessels, and internal organs. Type III collagen is common in fast growing tissue, particularly at the early stages of wound repair (19Haukipuro K. Risteli L. Kairaluoma M.I. Risteli J. Ann. Surg. 1987; 206: 752-756Crossref PubMed Scopus (53) Google Scholar, 20Liu X. Wu H. Byrne M. Krane S. Jaenisch R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1852-1865Crossref PubMed Scopus (437) Google Scholar). Mutant mice have been generated by gene targeting in which the type III collagen gene (COL3A1) has been knocked out (20Liu X. Wu H. Byrne M. Krane S. Jaenisch R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1852-1865Crossref PubMed Scopus (437) Google Scholar). These knock-out mice exhibit irregularly sized collagen fibers in the skin dermis as well as the aortic adventitia, which indicates the importance of type III collagen in regulating collagen fiber size. Notably, type III collagen null mice die perinatally, usually because of blood vessel or intestinal rupture. Thus, these data demonstrate that type III collagen is necessary for proper organ and tissue function, especially in distensible organs. These findings are consistent with the fact that mutations of type III collagen cause the most severe form of Ehlers-Danlos syndrome, EDS IV, 3The abbreviations used are: EDS, Ehlers-Danlos syndrome; MAD, multiwavelength anomalous diffraction; HPLC, high pressure liquid chromatography; Fmoc, N-(9-fluorenyl)methoxycarbonyl; ASU, asymmetric unit. which affect internal organs, arteries, joints, and skin. Indeed, EDS IV can result in sudden death when the large arteries rupture (20Liu X. Wu H. Byrne M. Krane S. Jaenisch R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1852-1865Crossref PubMed Scopus (437) Google Scholar). The most severe forms of EDS IV are correlated with point mutations that substitute a residue for a glycine near the C-terminal end of the triple helix, including G1012R, G1018V, and G1021E (21Smith L.T. Schwarze U. Goldstein J. Byers P.H. J. Investig. Dermatol. 1997; 108: 241-247Abstract Full Text PDF PubMed Scopus (67) Google Scholar). Structurally, type III collagen is a homotrimer composed of three α1(III) chains and resembles other fibrillar collagens. A key feature in the formation of type III collagen is a so-called disulfide or cystine knot, which is located between the triple helical region and the C-terminal telopeptide (22Bulleid N.J. Wilson R. Lees J.F. Biochem. J. 1996; 317: 195-202Crossref PubMed Scopus (62) Google Scholar, 23Allmann H. Fietzek P.P. Glanville R.W. Kühn K. Hoppe-Seyler's Z. Physiol. Chem. 1979; 360: 861-868PubMed Google Scholar). The knot is formed by three interchain disulfide bonds, and it significantly stabilizes the triple helical structure. The stability imparted by the disulfide knot has been successfully used to produce collagenous peptides that otherwise would be too unstable to study (24Mechling D.E. Bachinger H.P. J. Biol. Chem. 2000; 275: 14532-14536Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 25Boulègue C. Musiol H.J. Götz M.G. Renner C. Moroder L. Antioxid. Redox Signal. 2008; 10: 113-125Crossref PubMed Scopus (20) Google Scholar). Production of these peptides involves the C-terminal extension by the bis-cysteinyl-sequence GPCCG, followed by air or glutathione oxidation at lower temperature under slightly basic conditions. In the 1950s two structural models for collagen with different fiber periods and helical symmetries were proposed based on the fiber diffraction pattern of native collagen. These were the 7/2 helical model with a 20-Å axial repeat (26Cohen H. Br. J. Phys. Med. 1953; 16: 169-170PubMed Google Scholar, 27Cowan P.M. McGavin S. North A.C. Nature. 1955; 176: 1062-1064Crossref PubMed Scopus (116) Google Scholar) and the 10/3 helical model with a 30-Å axial repeat (27Cowan P.M. McGavin S. North A.C. Nature. 1955; 176: 1062-1064Crossref PubMed Scopus (116) Google Scholar). These single-helical models were discarded after the proposal of a triple helical structure with 10/3 helical symmetry and a 28.6-Å axial repeat by Ramachandran and Kartha (28Ramachandran G.N. Kartha G. Nature. 1955; 174: 269-270Crossref Scopus (258) Google Scholar). The first crystal structure of (Pro-Pro-Gly)10 showed a triple helix with 7/2 helical symmetry with a 20-Å axial repeat (29Okuyama K. Tanaka N. Ashida T. Kakudo M. Sakakibara S. J. Mol. Biol. 1972; 72: 571-576Crossref PubMed Scopus (53) Google Scholar). Recently, the fiber diffraction analysis of native collagen was performed based on the advanced diffraction data acquisition techniques and revealed that the x-ray diffraction data can be explained not only by the prevailing 10/3 helical model but also by the 7/2 helical model (7Okuyama K. Xu X. Iguchi M. Noguchi K. Biopolymers. 2006; 84: 181-191Crossref PubMed Scopus (75) Google Scholar). Almost all of the high resolution structures of model peptides adopt a 7/2 helical symmetry, and the conformation close to the 10/3 helix appears only in the guest region of host-guest peptides, like the T3-785 peptide and the integrin-binding protein complexed with integrin (7Okuyama K. Xu X. Iguchi M. Noguchi K. Biopolymers. 2006; 84: 181-191Crossref PubMed Scopus (75) Google Scholar). Crystallization and structure determination of collagen peptides is a challenging task. All of the available crystal structures are of either artificial mimics (like (GPP)n or (GPO)n, where O = 4(R)-hydroxyproline) or host-guest peptides where a short stretch of one to three native tripeptide units are flanked by three to five GPO repeats. Unfortunately, these structures give only limited insight into how side chains other than imino acid residues in the Xaa and Yaa positions contribute to collagen structure and stability (30Berisio R. Vitagliano L. Mazzarella L. Zagari A. 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Longer fragments impart higher flexibility, which may impede crystallization, whereas the production of a thermally stable triple helix actually requires longer sequences and the addition of artificial stabilizing tripeptide units, like GPO. These factors have limited the length of collagen fragments that have crystallized to only nine to eleven tripeptide units. Furthermore, this has restricted the number of integrated native tripeptide units to only one to three. One strategy used to obtain structural information on a longer triple helix was to fuse a stable trimeric molecule, foldon, which is a trimerization domain of fibritin, to a (GPP)10 collagen mimic (44Frank S. Jenny M. Schulthess T. Kammerer R.A. Boudko S. Landwehr R. Okuyama K. Engel J. Structure. 2003; 11: 339-346Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The resulting crystal structure revealed that there was a dramatic kink between the (GPP)10 triple helix and the foldon domain, which permitted adaptation to the mismatch between the 3-fold rotation symmetry of the foldon domain and the one residue stagger of the collagenous structure (45Frank S. Kammerer R.A. Mechling D. Schulthess T. Landwehr R. Bann J. Guo Y. Lustig A. Bächinger H.P. Engel J. J. Mol. Biol. 2001; 308: 1081-1089Crossref PubMed Scopus (166) Google Scholar). Here we describe another approach to stabilize a long collagen triple helix structure by the use of a native type III collagen C-terminal disulfide knot (46Frank S. Boudko S. Mizuno K. Schulthess T. Engel J. Bächinger H.P. J. Biol. Chem. 2003; 278: 7747-7750Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 47Barth D. Kyrieleis O. Frank S. Renner C. Moroder L. Chemistry. 2003; 9: 3703-3714Crossref PubMed Scopus (57) Google Scholar, 48Boudko S.P. Engel J. J. Mol. Biol. 2004; 335: 1289-1297Crossref PubMed Scopus (41) Google Scholar, 49Bachmann A. Kiefhaber T. Boudko S. Engel J. Bächinger H.P. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13897-13902Crossref PubMed Scopus (45) Google Scholar). Specifically, we crystallized a 42-residue collagen peptide containing the C-terminal type III collagen sequence from residues 991 to 1032, which contains the residues most commonly mutated in very severe forms of Ehlers-Danlos syndrome IV. This peptide also contains the native C-terminal cystine knot region. Thus, we demonstrate that utilization of the cystine knot opens doors to crystallization of lengthy and native collagen fragments. Peptide Sequences—The sequence of the type III collagen peptide used in this study is as follows: (GPI GPO GPR GNR GER GSE GSO GHO GsMO GPO GPO GAO GPCCGG)3.(Ois 4(R)-hydroxyproline, and sM is l-selenomethionine). These residues correspond to residues 991–1032 of the human collagen III a chain, with exception of the l-selenomethionine, which was substituted in place of the wild type glutamine for use in multiple wavelength anomalous diffraction (MAD) phasing. Peptide Synthesis and Purification—The peptide was synthesized on an ABI433A peptide synthesizer with 0.25 mm Fmoc-Gly-PEG-PS resin, a 4-fold excess of Fmoc amino acids and O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate as activating agent. The Fmoc amino acids carried the protection groups Cys-Trt, Hyp-t-Bu, Gln-Trt, His-Trt, Ser-t-Bu, Glu-O-t-Bu, Arg-Pbf, Asn-Trt, and Tyr-t-Bu. The peptide was cleaved off the resin and deprotected for 4 h at room temperature with 90% trifluoroacetic acid, 5% thioanisole, 3% 1,2-ethanedithiol, 2% anisole. Subsequently, the peptide was precipitated in cold ether, redissolved in H2O, and lyophilized. The reduced peptide was then purified by reverse phase HPLC using a C18 column (Vydac, Hesperia, CA; 50 × 250 mm, 10–15-μm particle size, 300-Å pores) with an acetonitrile/water gradient and 0.1% trifluoroacetic acid as ion-pairing agent. Finally, the peptide was characterized by electrospray/quadrupole/time-of-flight mass spectrometry (Q-tof micro; Waters Associates) and amino acid analysis. Peptide Folding, Oxidation, and Purification of Disulfide-linked Trimer—The lyophilized, reduced peptide was dissolved in degassed and N2 saturated 50 mm sodium acetate buffer, pH 4.5, under N2 atmosphere and was kept at 4 °C for 24 h to allow triple helix formation prior to oxidation. Two strategies of oxidation were used: exposure to atmospheric O2 or addition of reduced (10 mm) and oxidized (1 mm) glutathione and exposure to atmospheric O2. In both cases, the pH was raised to 8.3 with a saturated solution of Tris. Oxidation was carried out for 5–7 days, and the peptide mass was periodically analyzed by liquid chromatography-mass spectrometry. The maximum yield of covalently linked trimeric peptide was ∼60–70% and was slightly higher when glutathione was used for oxidation. To separate covalently linked trimeric peptide from other oligomers, the oxidized crude material was dissolved in deionized 8 m urea solution with 0.1% trifluoroacetic acid to prevent disulfide exchange and applied to a sieve column. Trimer-containing fractions were pooled out and further purified by reverse phase HPLC using a C18 column. Peptide Crystallization, Data Collection, Structure Determination, and Refinement—The purified and lyophilized covalently linked trimeric collagen III peptide, Gly991–Gly1032, was dissolved at a concentration of 15 mg/ml in 5 mm acetic acid. The peptide was crystallized at 22 °C using the hanging drop vapor diffusion method. For crystallization, 2 μl of the peptide solution was mixed with 2 μl of the reservoir solution of 20% polyethylene glycol monomethyl ether 550. The crystals appeared as very thin plates in a period of 1–5 days and are monoclinic, space group P21 with a = 31.98 Å, b = 21.52 Å, c = 68.97 Å, and β = 92.58°. Although the crystals diffracted beyond 2.5-Å resolution, they displayed extremely high mosaic spread (>3.0°) even when x-ray intensity data were collected at room temperature. Thus, several strategies for cryoprotection were tried in an attempt to improve the quality of the diffraction. The data were collected at ALS Beamline 8.2.1. Good quality data were obtained for only one crystal. For this data collection, glycerol was first added to the drop containing the crystal to a final concentration of 10%. The drop sat for 8 h, and the crystal was placed directly in the cryostream. However, at this point the mosaic spread was still unacceptably high. Thus, the crystal was annealed several times by removing the crystal from the cryostream and placing back in the drop solution. After two annealing cycles the diffraction, although still highly mosaic (1.7°), was of sufficient quality to collect data. A complete three wavelength MAD data set was collected on this crystal and was used for structure determination. The positions of the three selenomethionines in the triple helix were obtained using SOLVE. The phases obtained from these positions were improved by density modification in CNS, and the resulting density modified map was used for model building in O (50Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar, 51Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar, 52Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). After two-thirds of the structure was built, phase combination using phases from the partial model was used to improve the map. This permitted the current structure, which consists of one complete triple helix in the crystallographic asymmetric unit (ASU) to be built. Multiple cycles of simulated annealing, xyzb refinement, and rebuilding in O resulted in an Rwork/Rfree of 24.3%/27/4% to 2.30 Å resolution. The current model has excellent stereochemistry (Table 1) (53Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Multiple omit maps were calculated throughout the process to confirm the correctness of the model. Nonetheless, the electron density of the cystine knot residues remained poor, and the C-terminal residues that precede the cysteine residues display high B-factors consistent with this region being disordered or consisting of multiple conformations.TABLE 1Selected crystallographic data for Gly991–Gly1032 structureWavelength (λ)0.97971.02000.9796Resolution (Å)68.84-2.3068.78-2.3068.84-2.30Overall Rsym(%)aRsym = ΣΣ|Ihkl – Ihkl(j)|/ΣIhkl, where Ihkl(j) is the observed intensity and Ihkl is the final average value of intensity7.5 (24.6)bThe values in parentheses are for the highest resolution shell6.2 (24.5)7.6 (24.5)Overall I/σ(I)20.5 (2.5)20.4 (2.3)20.4 (2.5)No. of total reflections19,65719,64019,658No. of unique reflections396439623960Multiplicity5.05.05.0Overall figure of meritcFigure of merit = <|ΣP(α)eiα/ΣP(α)| >, where α is the phase and P(α) is the phase probability distribution0.490Crystal parametersSpace groupP21Cell parameters (Å)a = 31.98, b = 21.52, c = 68.97, β = 92.58°Resolution (Å)68.8-2.30Overall Rsym(%)aRsym = ΣΣ|Ihkl – Ihkl(j)|/ΣIhkl, where Ihkl(j) is the observed intensity and Ihkl is the final average value of intensity6.2(24.5)Overall I/σ(I)20.4 (2.3)No. of total reflections19640No. of unique reflections3962Refinement statisticsResolution (Å)68.8-2.30Rwork/Rfree(%)dRwork = Σ||Fobs| – |Fcalc||/Σ|Fobs| and Rfree = Σ||Fobs| – |Fcalc||/Σ|Fobs|, where all of the reflections belong to a test set of 5% randomly selected data24.3/27.4Root mean square deviationBond angles (°)1.92Bond lengths (Å)0.008B values (Å2)1.5Ramachandran analysisMost favored (%/no.)92.9/52Additionally allowed (%/no.)7.1/4Generously allowed (%/no.)0.0/0Disallowed (%/no.)0.0/0a Rsym = ΣΣ|Ihkl – Ihkl(j)|/ΣIhkl, where Ihkl(j) is the observed intensity and Ihkl is the final average value of intensityb The values in parentheses are for the highest resolution shellc Figure of merit = <|ΣP(α)eiα/ΣP(α)| >, where α is the phase and P(α) is the phase probability distributiond Rwork = Σ||Fobs| – |Fcalc||/Σ|Fobs| and Rfree = Σ||Fobs| – |Fcalc||/Σ|Fobs|, where all of the reflections belong to a test set of 5% randomly selected data Open table in a new tab Analysis of Triple Helix Geometry—Helical parameters were calculated based on the method of Sugeta and Miyazawa (54Sugeta H. Miyazawa T. Biopolymers. 1967; 5: 673-679Crossref Scopus (130) Google Scholar) for every amino acid residue using the program, PHEL (6Okuyama K. Wu G. Jiravanichanun N. Hongo C. Noguchi K. Biopolymers. 2006; 84: 421-432Crossref PubMed Scopus (49) Google Scholar). The input data for the calculation of the jth triplet consist of three sets of nine parameters for the jth, (j + 1)th, and (j + 2)th amino acid residues. Here, nine parameters of the jth amino acid residue are bond lengths of N(j)-Cα(j), Cα(j)-C′(j), and C′(j)-N(j + 1), bond angles of C′(j - 1)-N(j)-Cα(j), N(j)-Cα(j)-C′(j) and Cα(j)-C′(j)-N(j + 1), and dihedral angles of C′(j - 1)-N(j)-Cα(j)-C′(j), N(j)-Cα(j)-C′(j)-N(j + 1), and Cα(j)-C′(j)-N(j + 1)-Cα(j + 1). Folding and Oxidation of the Gly991–Gly1032 Peptide—Previous studies demonstrated that the corre
