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Collagen superfamily of proteins is a major component of the extracellular matrix. Defects in collagens underlie the cause of nearly 40 human genetic diseases in millions of people worldwide. Pathogenesis typically involves genetic alterations of the triple helix, a hallmark structural feature that bestows exceptional mechanical resistance to tensile forces and a capacity to bind a plethora of macromolecules. Yet, there is a paramount knowledge gap in understanding the functionality of distinct sites along the triple helix. Here, we present a recombinant technique to produce triple helical fragments for functional studies. The experimental strategy utilizes the unique capacity of the NC2 heterotrimerization domain of collagen IX to drive three α-chain selection and registering the triple helix stagger. For proof of principle, we produced and characterized long triple helical fragments of collagen IV that were expressed in a mammalian system. The heterotrimeric fragments encompassed the CB3 trimeric peptide of collagen IV, which harbors the binding motifs for α 1 β 1 and α 2 β 1 integrins. Fragments were characterized and shown to have a stable triple helix, post-translational modifications, and high affinity and specific binding of integrins. The NC2 technique is a universal tool for the high-yield production of heterotrimeric fragments of collagens. Fragments are suitable for mapping functional sites, determining coding sequences of binding sites, elucidating pathogenicity and pathogenic mechanisms of genetic mutations, and production of fragments for protein replacement therapy., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Collagens are the most abundant proteins in the body and among the most biosynthetically complex. A molecular ensemble of over 20 endoplasmic reticulum resident proteins participates in collagen biosynthesis and contributes to heterogeneous post-translational modifications. Pathogenic variants in genes encoding collagens cause connective tissue disorders, including osteogenesis imperfecta, Ehlers-Danlos syndrome, and Gould syndrome (caused by mutations in COL4A1 and COL4A2), and pathogenic variants in genes encoding proteins required for collagen biosynthesis can cause similar but overlapping clinical phenotypes. Notably, pathogenic variants in lysyl hydroxylase 3 (LH3) cause a multisystem connective tissue disorder that exhibits pathophysiological features of collagen-related disorders. LH3 is a multifunctional collagen-modifying enzyme; however, its precise role(s) and substrate specificity during collagen biosynthesis has not been defined. To address this critical gap in knowledge, we generated LH3 KO cells and performed detailed quantitative and molecular analyses of collagen substrates. We found that LH3 deficiency severely impaired secretion of collagen α1α1α2(IV) but not collagens α1α1α2(I) or α1α1α1(III). Amino acid analysis revealed that LH3 is a selective LH for collagen α1α1α2(IV) but a general glucosyltransferase for collagens α1α1α2(IV), α1α1α2(I), and α1α1α1(III). Importantly, we identified rare variants that are predicted to be pathogenic in the gene encoding LH3 in two of 113 fetuses with intracranial hemorrhage-a cardinal feature of Gould syndrome. Collectively, our findings highlight a critical role of LH3 in α1α1α2(IV) biosynthesis and suggest that LH3 pathogenic variants might contribute to Gould syndrome., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Collagen-derived hydroxyproline (Hyp)-containing peptides have a variety of biological effects on cells. These bioactive collagen peptides are locally generated by the degradation of endogenous collagen in response to injury. However, no comprehensive study has yet explored the functional links between Hyp-containing peptides and cellular behavior. Here, we show that the dipeptide prolyl-4-hydroxyproline (Pro-Hyp) exhibits pronounced effects on mouse tendon cells. Pro-Hyp promotes differentiation/maturation of tendon cells with modulation of lineage-specific factors and induces significant chemotactic activity in vitro. In addition, Pro-Hyp has profound effects on cell proliferation, with significantly upregulated extracellular signal-regulated kinase phosphorylation and extracellular matrix production and increased type I collagen network organization. Using proteomics, we have predicted molecular transport, cellular assembly and organization, and cellular movement as potential linked-network pathways that could be altered in response to Pro-Hyp. Mechanistically, cells treated with Pro-Hyp demonstrate increased directional persistence and significantly increased directed motility and migration velocity. They are accompanied by elongated lamellipodial protrusions with increased levels of active β1-integrin-containing focal contacts, as well as reorganization of thicker peripheral F-actin fibrils. Pro-Hyp-mediated chemotactic activity is significantly reduced (p < 0.001) in cells treated with the mitogen-activated protein kinase kinase 1/2 inhibitor PD98059 or the α5β1-integrin antagonist ATN-161. Furthermore, ATN-161 significantly inhibits uptake of Pro-Hyp into adult tenocytes. Thus, our findings document the molecular basis of the functional benefits of the Pro-Hyp dipeptide in cellular behavior. These dynamic properties of collagen-derived Pro-Hyp dipeptide could lead the way to its application in translational medicine., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Osteogenesis imperfecta (OI) is a heritable brittle bone disease mainly caused by mutations in the two type I collagen genes. Collagen synthesis is a complex process including trimer formation, glycosylation, secretion, extracellular matrix (ECM) formation, and mineralization. Using OI patient-derived fibroblasts and induced pluripotent stem cells (iPSCs), we investigated the effect of 4-phenylbutyric acid (4-PBA) on collagen synthesis to test its potential as a new treatment for OI. Endoplasmic reticulum (ER) retention of type I collagen was observed by immunofluorescence staining in OI patient-derived fibroblasts with glycine substitution and exon skipping mutations. Liquid chromatography-mass spectrometry analysis revealed excessive glycosylation of secreted type I collagen at the specific sites in OI cells. The misfolding of the type I collagen triple helix in the ECM was demonstrated by the incorporation of heat-dissociated collagen hybridizing peptide in OI cells. Type I collagen was produced excessively by OI fibroblasts with a glycine mutation, but this excessive production was normalized when OI fibroblasts were cultured on control fibroblast-derived ECM. We also found that mineralization was impaired in osteoblasts differentiated from OI iPSCs. In summary, treatment with 4-PBA normalizes the excessive production of type I collagen, reduces ER retention, partially improves misfolding of the type I collagen helix in ECM, and improves osteoblast mineralization. Thus, 4-PBA may improve not only ER retention, but also type I collagen synthesis and mineralization in human cells from OI patients., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Collagen is the most abundant protein in humans. It has a characteristic triple-helix structure and is heavily posttranslationally modified. The complex biosynthesis of collagen involves processing by many enzymes and chaperones in the rough endoplasmic reticulum. Lysyl hydroxylase 1 (LH1) is required to hydroxylate lysine for cross-linking and carbohydrate attachment within collagen triple helical sequences. Additionally, a recent study of prolyl 3-hydroxylase 3 (P3H3) demonstrated that this enzyme may be critical for LH1 activity; however, the details surrounding its involvement remain unclear. If P3H3 is an LH1 chaperone that is critical for LH1 activity, P3H3 and LH1 null mice should display a similar deficiency in lysyl hydroxylation. To test this hypothesis, we compared the amount and location of hydroxylysine in the triple helical domains of type V and I collagen from P3H3 null, LH1 null, and wild-type mice. The amount of hydroxylysine in type V collagen was reduced in P3H3 null mice, but surprisingly type V collagen from LH1 null mice contained as much hydroxylysine as type V collagen from wild-type mice. In type I collagen, our results indicate that LH1 plays a global enzymatic role in lysyl hydroxylation. P3H3 is also involved in lysyl hydroxylation, particularly at cross-link formation sites, but is not required for all lysyl hydroxylation sites. In summary, our study suggests that LH1 and P3H3 likely have two distinct mechanisms to recognize different collagen types and to distinguish cross-link formation sites from other sites in type I collagen., Competing Interests: Conflict of interest The authors declare that they have no competing interests related to this work., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Extracellular matrix proteins are biosynthesized in the rough endoplasmic reticulum (rER), and the triple-helical protein collagen is the most abundant extracellular matrix component in the human body. Many enzymes, molecular chaperones, and post-translational modifiers facilitate collagen biosynthesis. Collagen contains a large number of proline residues, so the cis / trans isomerization of proline peptide bonds is the rate-limiting step during triple-helix formation. Accordingly, the rER-resident peptidyl prolyl cis / trans isomerases (PPIases) play an important role in the zipper-like triple-helix formation in collagen. We previously described this process as "Ziploc-ing the structure" and now provide additional information on the activity of individual rER PPIases. We investigated the substrate preferences of these PPIases in vitro using type III collagen, the unhydroxylated quarter fragment of type III collagen, and synthetic peptides as substrates. We observed changes in activity of six rER-resident PPIases, cyclophilin B (encoded by the PPIB gene), FKBP13 ( FKBP2 ), FKBP19 ( FKBP11 ), FKBP22 ( FKBP14 ), FKBP23 ( FKBP7 ), and FKBP65 ( FKBP10 ), due to posttranslational modifications of proline residues in the substrate. Cyclophilin B and FKBP13 exhibited much lower activity toward post-translationally modified substrates. In contrast, FKBP19, FKBP22, and FKBP65 showed increased activity toward hydroxyproline-containing peptide substrates. Moreover, FKBP22 showed a hydroxyproline-dependent effect by increasing the amount of refolded type III collagen in vitro and FKBP19 seems to interact with triple helical type I collagen. Therefore, we propose that hydroxyproline modulates the rate of Ziploc-ing of the triple helix of collagen in the rER., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Type I collagen extracted from tendon, skin, and bone of wild type and prolyl 3-hydroxylase 1 (P3H1) null mice shows distinct patterns of 3-hydroxylation and glycosylation of hydroxylysine residues. The A1 site (Pro-986) in the α1-chain of type I collagen is almost completely 3-hydroxylated in every tissue of the wild type mice. In contrast, no 3-hydroxylation of this proline residue was found in P3H1 null mice. Partial 3-hydroxylation of the A3 site (Pro-707) was present in tendon and bone, but absent in skin in both α-chains of the wild type animals. Type I collagen extracted from bone of P3H1 null mice shows a large reduction in 3-hydroxylation of the A3 site in both α-chains, whereas type I collagen extracted from tendon of P3H1 null mice shows little difference as compared with wild type. These results demonstrate that the A1 site in type I collagen is exclusively 3-hydroxylated by P3H1, and presumably, this enzyme is required for the 3-hydroxylation of the A3 site of both α-chains in bone but not in tendon. The increase in glycosylation of hydroxylysine in P3H1 null mice in bone was found to be due to an increased occupancy of normally glycosylated sites. Despite the severe disorganization of collagen fibrils in adult tissues, the D-period of the fibrils is unchanged. Tendon fibrils of newborn P3H1 null mice are well organized with only a slight increase in diameter. The absence of 3-hydroxyproline and/or the increased glycosylation of hydroxylysine in type I collagen disturbs the lateral growth of the fibrils.

Vascular Ehlers-Danlos syndrome (EDS) type IV is the most severe form of EDS. In many cases the disease is caused by a point mutation of Gly in type III collagen. A slower folding of the collagen helix is a potential cause for over-modifications. However, little is known about the rate of folding of type III collagen in patients with EDS. To understand the molecular mechanism of the effect of mutations, a system was developed for bacterial production of homotrimeric model polypeptides. The C-terminal quarter, 252 residues, of the natural human type III collagen was attached to (GPP)7 with the type XIX collagen trimerization domain (NC2). The natural collagen domain forms a triple helical structure without 4-hydroxylation of proline at a low temperature. At 33 °C, the natural collagenous part is denatured, but the C-terminal (GPP)7-NC2 remains intact. Switching to a low temperature triggers the folding of the type III collagen domain in a zipper-like fashion that resembles the natural process. We used this system for the two known EDS mutations (Gly-to-Val) in the middle at Gly-910 and at the C terminus at Gly-1018. In addition, wild-type and Gly-to-Ala mutants were made. The mutations significantly slow down the overall rate of triple helix formation. The effect of the Gly-to-Val mutation is much more severe compared with Gly-to-Ala. This is the first report on the folding of collagen with EDS mutations, which demonstrates local delays in the triple helix propagation around the mutated residue.

The rate-limiting step of folding of the collagen triple helix is catalyzed by cyclophilin B (CypB). The G6R mutation in cyclophilin B found in the American Quarter Horse leads to autosomal recessive hyperelastosis cutis, also known as hereditary equine regional dermal asthenia. The mutant protein shows small structural changes in the region of the mutation at the side opposite the catalytic domain of CypB. The peptidylprolyl cis-trans isomerase activity of the mutant CypB is normal when analyzed in vitro. However, the biosynthesis of type I collagen in affected horse fibroblasts shows a delay in folding and secretion and a decrease in hydroxylysine and glucosyl-galactosyl hydroxylysine. This leads to changes in the structure of collagen fibrils in tendon, similar to those observed in P3H1 null mice. In contrast to cyclophilin B null mice, where little 3-hydroxylation was found in type I collagen, 3-hydroxylation of type I collagen in affected horses is normal. The mutation disrupts the interaction of cyclophilin B with the P-domain of calreticulin, with lysyl hydroxylase 1, and probably other proteins, such as the formation of the P3H1·CypB·cartilage-associated protein complex, resulting in less effective catalysis of the rate-limiting step in collagen folding in the rough endoplasmic reticulum.

Osteogenesis imperfecta (OI) is a skeletal disorder primarily caused by mutations in the type I collagen genes. However, recent investigations have revealed that mutations in the genes encoding for cartilage-associated protein (CRTAP) or prolyl 3-hydroxylase 1 (P3H1) can cause a severe, recessive form of OI. These reports show minimal 3-hydroxylation of key proline residues in type I collagen as a result of CRTAP or P3H1 deficiency and demonstrate the importance of P3H1 and CRTAP to bone structure and development. P3H1 and CRTAP have previously been shown to form a stable complex with cyclophilin B, and P3H1 was shown to catalyze the 3-hydroxylation of specific proline residues in procollagen I in vitro. Here we describe a mouse model in which the P3H1 gene has been inactivated. Our data demonstrate abnormalities in collagen fibril ultrastructure in tendons from P3H1 null mice by electron microscopy. Differences are also seen in skin architecture, as well as in developing limbs by histology. Additionally bone mass and strength were significantly lower in the P3H1 mice as compared with wild-type littermates. Altogether these investigations demonstrate disturbances of collagen fiber architecture in tissues rich in fibrillar collagen, including bone, tendon, and skin. This model system presents a good opportunity to study the underlying mechanisms of recessive OI and to better understand its effects in humans.

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 2.3A resolution. This represents the longest collagen triple helical structure determined to date with a native sequence. Strikingly, the Gly(991)-Gly(1032) 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.

Collagen has a triple helical structure comprising strands with a repeating Xaa-Yaa-Gly sequence. L-Proline (Pro) and 4(R)-hydroxyl-L-proline (4(R)Hyp) residues are found most frequently in the Xaa and Yaa positions. However, in natural collagen, 3(S)-hydroxyl-L-proline (3(S)Hyp) occurs in the Xaa positions to varying extents and is most common in collagen types IV and V. Although 4(R)Hyp residues in the Yaa positions have been shown to be critical for the formation of a stable triple helix, the role of 3(S)Hyp residues in the Xaa position is not well understood. Indeed, recent studies have demonstrated that the presence of 3(S)Hyp in the Xaa positions of collagen-like peptides actually has a destabilizing effect relative to peptides with Pro in these locations. Whether this destabilization is reflected in a local unfolding or in other structural alterations of the collagen triple helix is unknown. Thus, to determine what effect the presence of 3(S)Hyp residues in the Xaa positions has on the overall conformation of the collagen triple helix, we determined the crystal structure of the polypeptide H-(Gly-Pro-4(R)Hyp)3-(Gly-3(S)Hyp-4(R)Hyp)2-(Gly-Pro-4(R)Hyp)4-OH to 1.80 A resolution. The structure shows that, despite the presence of the 3(S)Hyp residues, the peptide still adopts a typical 7/2 superhelical symmetry similar to that observed in other collagen structures. The puckering of the Xaa position 3(S)Hyp residues, which are all down (Cgamma-endo), and the varphi/psi dihedral angles of the Xaa 3(S)Hyp residues are also similar to those of typical collagen Pro Xaa residues. Thus, the presence of 3(S)Hyp in the Xaa positions does not lead to large structural alterations in the collagen triple helix.

The collagen triple helix is characterized by the repeating sequence motif Gly-Xaa-Yaa, where Xaa and Yaa are typically proline and (2S,4R)-4-hydroxyproline (4(R)Hyp), respectively. Previous analyses have revealed that H-(Pro-4(R)Hyp-Gly)(10)-OH forms a stable triple helix, whereas H-(4(R)Hyp-Pro-Gly)(10)-OH does not. Several theories have been put forth to explain the importance of proline puckering and conformation in triple helix formation; however, the details of how they affect triple helix stability are unknown. Underscoring this, we recently demonstrated that the polypeptide Ac-(Gly-4(R)Hyp-4(R)Hyp)(10)-NH(2) forms a triple helix that is more stable than Ac-(Gly-Pro-4(R)Hyp)(10)-NH(2). Here we report crystal the structure of the H-(Gly-4(R)Hyp-4(R)Hyp)(9)-OH peptide at 1.55 A resolution. The puckering of the Yaa position 4(R)Hyp in this structure is up (Cgamma exo), as has been found in other collagen peptide structures. Notably, however, the 4(R)Hyp in the Xaa position also takes the up pucker, which is distinct from all other collagen structures. Regardless of the notable difference in the Xaa proline puckering, our structure still adopts a 7/2 superhelical symmetry similar to that observed in other collagen structures. Thus, the basis for the observed differences in the thermodynamic data of the triple helix<--> coil transition between our peptide and other triple helical peptides likely results from contributions from the unfolded state. Indeed, the unfolded state of the H-(Gly-4(R)Hyp-4(R)Hyp)(9)-OH peptide seems to be stabilized by a preformed polyproline II helix in each strand, which could be explained by the presence of a unique repeating intra-strand water-mediated bridge observed in the H-(Gly-4(R)Hyp-4(R)Hyp)(9)-OH structure, as well as a higher amount of trans peptide bonds.

The collagen triple helix is one of the most abundant protein motifs in animals. The structural motif of collagen is the triple helix formed by the repeated sequence of -Gly-Xaa-Yaa-. Previous reports showed that H-(Pro-4(R)Hyp-Gly)(10)-OH (where '4(R)Hyp' is (2S,4R)-4-hydroxyproline) forms a trimeric structure, whereas H-(4(R)Hyp-Pro-Gly)(10)-OH does not form a triple helix. Compared with H-(Pro-Pro-Gly)(10)-OH, the melting temperature of H-(Pro-4(R)Hyp-Gly)(10)-OH is higher, suggesting that 4(R)Hyp in the Yaa position has a stabilizing effect. The inability of triple helix formation of H-(4(R)Hyp-Pro-Gly)(10)-OH has been explained by a stereoelectronic effect, but the details are unknown. In this study, we synthesized a peptide that contains 4(R)Hyp in both the Xaa and the Yaa positions, that is, Ac-(Gly-4(R)Hyp-4(R)Hyp)(10)-NH(2) and compared it to Ac-(Gly-Pro-4(R)Hyp)(10)-NH(2), and Ac-(Gly-4(R)Hyp-Pro)(10)-NH(2). Ac-(Gly-4(R)Hyp-4(R)Hyp)(10)-NH(2) showed a polyproline II-like circular dichroic spectrum in water. The thermal transition temperatures measured by circular dichroism and differential scanning calorimetry were slightly higher than the values measured for Ac-(Gly-Pro-4(R)Hyp)(10)-NH(2) under the same conditions. For Ac-(Gly-4(R)Hyp-4(R)Hyp)(10)-NH(2), the calorimetric and the van't Hoff transition enthalpy DeltaH were significantly smaller than that of Ac-(Gly-Pro-4(R)Hyp)(10)-NH(2). We postulate that the denatured states of the two peptides are significantly different, with Ac-(Gly-4(R)Hyp-4(R)Hyp)(10)-NH(2) forming a more polyproline II-like structure instead of a random coil. Two-dimensional nuclear Overhauser effect spectroscopy suggests that the triple helical structure of Ac-(Gly-4(R)Hyp-4(R)Hyp)(10)-NH(2) is more flexible than that of Ac-(Gly-Pro-4(R)Hyp)(10)-NH(2). This is confirmed by the kinetics of amide (1)H exchange with solvent deuterium of Ac-(Gly-4(R)Hyp-4(R)Hyp)(10)-NH(2), which is faster than that of Ac-(Gly-Pro-4(R)Hyp)(10)-NH(2). The higher transition temperature of Ac-(Gly-4(R)Hyp-4(R)Hyp)(10)-NH(2), can be explained by the higher trans/cis ratio of the Gly-4(R)Hyp peptide bonds than that of the Gly-Pro bonds, and this ratio compensates for the weaker interchain hydrogen bonds.

Escherichia coli apomanganese superoxide dismutase, prepared by removing the native metal ion under denaturing conditions, exhibits thermally triggered metal uptake behavior previously observed for thermophilic and hyperthermophilic superoxide dismutases but over a lower temperature range. Differential scanning calorimetry of aposuperoxide dismutase and metalated superoxide dismutase unfolding transitions has provided quantitative estimates of the metal binding affinities for manganese superoxide dismutase. The binding constant for Mn(II) (K(Mn(II)) = 3.2 x 10(8) m(-1)) is surprisingly low in light of the essentially irreversible metal binding characteristic of this family of proteins and indicates that metal binding and release processes are dominated by kinetic, rather than thermodynamic, constraints. The kinetic stability of the metalloprotein complex can be traced to stabilization by elements of the protein that are independent of the presence or absence of the metal ion reflected in the thermally triggered metalation characteristic of these proteins. Binding constants for Mn(III), Fe(II), and Fe(III) complexes were estimated using quasireversible values for the unfolding enthalpy and DeltaC(p) for apo-Mn superoxide dismutase and the observed T(m) values for unfolding the metalated species in the absence of denaturants. For manganese and iron complexes, an oxidation state-dependent binding affinity reflects the protein perturbation of the metal redox potential.

Hydroxylation of proline residues in the Yaa position of the Gly-Xaa-Yaa repeated sequence to 4(R)-hydroxyproline is essential for the formation of the collagen triple helix. A small number of 3(S)-hydroxyproline residues are present in most collagens in the Xaa position. Neither the structural nor a biological role is known for 3(S)-hydroxyproline. To characterize the structural role of 3(S)-hydroxyproline, the peptide Ac-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 was synthesized and analyzed by circular dichroism spectroscopy, analytical ultracentrifugation, and 1H nuclear magnetic resonance spectroscopy. At 4 degrees C in water the circular dichroism spectrum indicates that this peptide was in a polyproline-II-like secondary structure with a positive peak at 225 nm similar to Ac-(Gly-Pro-4(R)Hyp)10-NH2. The positive peak at 225 nm almost linearly decreases with increasing temperature to 95 degrees C without an obvious transition. Although the peptide Ac-(Gly-Pro-4(R)Hyp)10-NH2 forms a trimer at 10 degrees C, sedimentation equilibrium experiments indicate that Ac-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 is a monomer in water at 7 degrees C. To study the role of 3(S)-hydroxyproline in the Yaa position, we synthesized Ac-(Gly-Pro-3(S)Hyp)10-NH2. This peptide also does not form a triple helix in water. 1H Nuclear magnetic resonance spectroscopy data (including line widths and nuclear Overhauser effects) are entirely consistent, with neither Ac-(Gly-3(S)Hyp-4(R)Hyp)10-NH2 nor Ac-(Gly-Pro-3(S)Hyp)10-NH2 forming a triple helix in water. Therefore 3(S)-hydroxyproline destabilizes the collagen triple helix in either position. In contrast, when 3(S)-hydroxyproline is inserted as a guest in the highly stable -Gly-Pro-4(R)Hyperepeated host sequence, Ac-(Gly-Pro-4(R)Hyp)3-Gly-3(S)Hyp-4(R)Hyp-(Gly-Pro-4(R)Hyp)4-Gly-Gly-NH2 forms as stable a trimer (Tm=49.6 degrees C) as Ac-(Gly-Pro-4(R)Hyp)8-Gly-Gly-NH2 (Tm=48.9 degrees C). Given that Ac-(Gly-Pro-4(R)Hyp)3-Gly-4(R)Hyp-Pro-(Gly-Pro-4(R)Hyp)4-Gly-Gly-NH2 forms a triple helix nearly as stable as the above two peptides (Tm=45.0 degrees C) and the knowledge that Ac-(Gly-4(R)Hyp-Pro)10-NH2 does not form a triple helix, we conclude that the host environment dominates the structure of host-guest peptides and that these peptides are not necessarily accurate predictors of triple helical stability.

4(R)-Hydroxyproline in the Yaa position of the -Gly-Xaa-Yaa-repeated sequence of collagen plays a crucial role in the stability of the triple helix. Since the peptide (4(R)-Hyp-Pro-Gly)10 does not form a triple helix, it was generally believed that polypeptides with a -Gly-4(R)-Hyp-Yaa-repeated sequence do not form a triple helix. Recently, we found that acetyl-(Gly-4(R)-Hyp-Thr)10-NH2 forms a triple helix in aqueous solutions. To further study the role of 4(R)-hydroxyproline in the Xaa position, we made a series of acetyl-(Gly-4(R)-Hyp-Yaa)10-NH2 peptides where Yaa was alanine, serine, valine, and allo-threonine. We previously hypothesized that the hydroxyl group of threonine might form a hydrogen bond to the hydroxyl group of 4(R)hydroxyproline. In water, only the threonine- and the valine-containing peptides were triple helical. The remaining peptides did not form a triple helix in water. In 1,2- and in 1,3-propanediol at 4 degrees C, all the soluble peptides were triple helical. From the transition temperature of the triple helices, it was found that among the examined residues, threonine was the most stable residue in the acetyl-(Gly-4(R)-Hyp-Yaa)10-NH2 peptide. The transition temperatures of the valine- and allo-threonine-containing peptides were 10 degrees lower than those of the threonine peptide. Surprisingly, the serine-containing peptide was the least stable. These results indicate that the stability of these peptides depends on the presence of a methyl group as well as the hydroxyl group and that the stereo configuration of the two groups is essential for the stability. In the threonine peptide, we hypothesize that the methyl group shields the interchain hydrogen bond between the glycine and the Xaa residue from water and that the hydroxyl groups of threonine and 4(R)hydroxyproline can form direct or water-mediated hydrogen bonds.

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 NH(2) 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 NH(2)- 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.