Design, expression and characterization of collagen-like proteins based on the cell adhesive and crosslinking sequences derived from native collagens

Two recombinant collagen-like proteins consisting of cell adhesion domains derived from native type I collagen were designed and synthesized by a genetic engineering method. The cross-linking sequence, GPPGPCCGGG, derived from collagen III was used to promote triple helix formation through the disulfide bonds formed among three chains by flanking the peptide at the C-terminal of the collagen-like proteins. SDS-PAGE and western-blotting data suggested possibility of the formation of a triple helix structure for both recombinant proteins. CD spectra and thermal stability analyses indicated that the triple-helix structure in the collagen-like proteins was pH-dependent and stabilized under acidic environmental condition. Moreover, the collagen-like protein flanked with the cross-linking sequence at the C-terminal showed the most stable triple-helical conformation under acidic conditions.     

Evidence for a type-IV-related collagen in Drosophila melanogaster. Evolutionary constancy of the carboxyl-terminal noncollagenous domain

Type IV collagen, a major structural component of basement membrane, has been characterized only in vertebrates. It is unique among the collagenous proteins in that it forms specific lattice networks by end-to-end interactions. In particular, in mammals the C-terminal noncollagenous domain (NCl) of collagen IV was shown to be one of the major cross-linking sites in the network assembly. Here, we give the first direct evidence of type-IV-related collagen in invertebrates by sequence analysis of cDNA and genomic DNA clones for the 3'-end of a previously characterized Drosophila collagen gene. The data describe the C-terminal 190 amino acid residues of the triple helix and the entire noncollagenous domain (231 amino acids) of the chain encoded for by this gene. Comparison with data reported for human and mouse alpha 1(IV) chains reveals that triple-helix regions are quite different, while NC1 structures are very similar. This suggests different constraints on triple-helix and NC1 domains during evolution. Present data support the assumption that the NC1 structure originated from duplication of an ancestral sequence; the extent of both interspecies and intramolecular homologies suggests the maintenance in vertebrates and invertebrates of an ancestral specific function.     

Osteogenesis Imperfecta Model Peptides: Incorporation of Residues Replacing Gly within a Triple Helix Achieved by Renucleation and Local Flexibility

Missense mutations, which replace one Gly with a larger residue in the repeating sequence of the type I collagen triple helix, lead to the hereditary bone disorder osteogenesis imperfecta (OI). Previous studies suggest that these mutations may interfere with triple-helix folding. NMR was used to investigate triple-helix formation in a series of model peptides where the residue replacing Gly, as well as the local sequence environment, was varied. NMR measurement of translational diffusion coefficients allowed the identification of partially folded species. When Gly was replaced by Ala, the Ala residue was incorporated into a fully folded triple helix, whereas replacement of Gly by Ser or Arg resulted in the presence of some partially folded species, suggesting a folding barrier. Increasing the triple-helix stability of the sequence N-terminal to a Gly-to-Ser replacement allowed complete triple-helix folding, whereas with the substitution of Arg, with its large side chain, the peptide achieved full folding only after flexible residues were introduced N-terminal to the mutation site. These studies shed light on the factors important for accommodation of Gly mutations within the triple helix and may relate to the varying severity of OI.     

Structure formation in the C terminus of type III collagen guides disulfide cross-linking

In type III collagen the main triple-helical domain is followed by a disulfide knot and the C-terminal propeptide, which are both essential for nucleation, stabilization and registration of the triple helix. We demonstrate that oxidative inter-chain disulfide bridging does not occur between the knot sequences GlyProCysCysGly of dissociated randomly coiled chains. N-terminal fusion of the obligatory trimeric domain of mini-fibritin is able to direct this process efficiently, demonstrating a folded precursor mechanism in which the thiol groups have to be properly placed for the formation of native disulfide bonds. The natural C-propeptide domain may act in a similar way as the mini-fibritin domain. After disulfide linkage and triple-helix formation the catalyzing mini-fibritin domain was removed by thrombin cleavage. In this way a short but stable triple-helical collagen fragment was expressed in Escherichia coli for structural and functional studies.     

Folding delay and structural perturbations caused by type IV collagen natural interruptions and nearby Gly missense mutations

The standard collagen triple helix requires Gly as every third residue in the amino acid sequence, yet all nonfibrillar collagens contain sites where this repeating pattern is interrupted. To explore the effects of such natural interruptions on the triple helix, a 4- or 15-residue sequence from human basement membrane type IV collagen was introduced between (Gly-Xaa-Yaa)(n) domains within a recombinant bacterial collagen. The interruptions had little effect on melting temperature, consistent with the high thermal stability reported for nonfibrillar collagens. Although the 4-residue interruption cannot be accommodated within a standard triple helix, trypsin and thermolysin resistance indicated a tightly packed structure. Central residues of the 15-residue interruption were protease-susceptible, whereas residues near the (Gly-Xaa-Yaa)(n) boundary were resistant, supporting a transition from an alternate conformation to a well packed triple helix. Both interruptions led to a delay in triple-helix folding, with the 15-residue interruption causing slower folding than the 4-residue interruption. These results suggest that propagation through interruptions represents a slow folding step. To clarify the relation between natural interruptions and pathological mutations, a Gly to Ser missense mutation was placed three triplets away from the 4-residue interruption. As a result of this mutation, the 4-residue interruption and nearby triple helix became susceptible to protease digestion, and an additional folding delay was observed. Because Gly missense mutations that cause disease are often located near natural interruptions, structural and folding perturbations arising from such proximity could be a factor in collagen genetic diseases.     

Mapping the collagen-binding site in the von Willebrand factor-A3 domain

The multimeric glycoprotein von Willebrand factor (VWF) mediates platelet adhesion to collagen at sites of vascular damage. The binding site for collagen types I and III is located in the VWF-A3 domain. Recently, we showed that His(1023), located near the edge between the "front" and "bottom" faces of A3, is critical for collagen binding (Romijn, R. A., Bouma, B., Wuyster, W., Gros, P., Kroon, J., Sixma, J. J., and Huizinga, E. G. (2001) J. Biol. Chem. 276, 9985-9991). To map the binding site in detail, we introduced 22 point mutations in the front and bottom faces of A3. The mutants were expressed as multimeric VWF, and binding to collagen type III was evaluated in a solid-state binding assay and by surface plasmon resonance. Mutation of residues Asp(979), Ser(1020), and His(1023) nearly abolished collagen binding, whereas mutation of residues Ile(975), Thr(977), Val(997), and Glu(1001) reduced binding affinity about 10-fold. Together, these residues define a flat and rather hydrophobic collagen-binding site located at the front face of the A3 domain. The collagen-binding site of VWF-A3 is distinctly different from that of the homologous integrin alpha(2) I domain, which has a hydrophilic binding site located at the top face of the domain. Based on the surface characteristics of the collagen-binding site of A3, we propose that it interacts with collagen sequences containing positively charged and hydrophobic residues. Docking of a collagen triple helix on the binding site suggests a range of possible engagements and predicts that at most eight consecutive residues in a collagen triple helix interact with A3.     

Defining requirements for collagenase cleavage in collagen type III using a bacterial collagen system

Degradation of fibrillar collagens is important in many physiological and pathological events. These collagens are resistant to most proteases due to the tightly packed triple-helical structure, but are readily cleaved at a specific site by collagenases, selected members of the matrix metalloproteinases (MMPs). To investigate the structural requirements for collagenolysis, varying numbers of GXY triplets from human type III collagen around the collagenase cleavage site were inserted between two triple helix domains of the Scl2 bacterial collagen protein. The original bacterial CL domain was not cleaved by MMP-1 (collagenase 1) or MMP-13 (collagenase 3). The minimum type III sequence necessary for cleavage by the two collagenases was 5 GXY triplets, including 4 residues before and 11 residues after the cleavage site (P4-P11'). Cleavage of these chimeric substrates was not achieved by the catalytic domain of MMP-1 or MMP-13, nor by full-length MMP-3. Kinetic analysis of the chimeras indicated that the rate of cleavage by MMP-1 of the chimera containing six triplets (P7-P11') of collagen III was similar to that of native collagen III. The collagenase-susceptible chimeras were cleaved very slowly by trypsin, a property also seen for native collagen III, supporting a local structural relaxation of the triple helix near the collagenase cleavage site. The recombinant bacterial-human collagen system characterized here is a good model to investigate the specificity and mechanism of action of collagenases.     

Interaction of collagen-like peptide models of asymmetric acetylcholinesterase with glycosaminoglycans: spectroscopic studies of conformational changes and stability

The effect of heparin on the conformation and stability of triple-helical peptide models of the collagen tail of asymmetric acetylcholinesterase expands our understanding of heparin interactions with proteins and presents an opportunity for clarifying the nature of binding of ligands to collagen triple-helix domains. Within the collagen tail of AChE, there are two consensus sequences for heparin binding of the form BBXB, surrounded by additional basic residues. Circular dichroism studies were used to determine the effect of the addition of increasing concentrations of heparin on triple-helical peptide models for the heparin binding domains, including peptides in which the basic residues within and surrounding the consensus sequence were replaced by alanine residues. The addition of heparin caused an increased triple-helix content with saturation properties for the peptide modeling the C-terminal site, while precipitation, with no increased helix content resulted from heparin addition to the peptide modeling the N-terminal site. The results suggest that the two binding sites with a similar triple-helical conformation have distinctive ways of interacting with heparin, which must relate to small differences in the consensus sequence (GRKGR vs GKRGK) and in the surrounding basic residues. Addition of heparin increased the thermal stability of all peptides containing the consensus sequence. Heparan sulfate produced conformational and stabilization effects similar to those of heparin, while chondroitin sulfate led to a cloudy solution, loss of circular dichroism signal, and a smaller increase in thermal stability. Thus, specificity in both the sequence of the triple helix and the type of glycosaminoglycan is required for this interaction.     

Common interruptions in the repeating tripeptide sequence of non-fibrillar collagens: sequence analysis and structural studies on triple-helix peptide models

Interruptions in the repeating (Gly-X1-X2)_n amino acid sequence pattern are found in the triple-helix domains of all non-fibrillar collagens, and perturbations to the triple-helix at such sites are likely to play a role in collagen higher-order structure and function. This study defines the sequence features and structural consequences of the most common interruption, where one residue is missing from the tripeptide pattern, Gly-X1-X2-Gly-AA₁-Gly-X1-X2, designated G1G interruptions. Residues found within G1G interruptions are predominantly hydrophobic (70%), followed by a significant amount of charged residues (16%), and the Gly-X1-X2 triplets flanking the interruption are atypical. Studies on peptide models indicate the degree of destabilization is much greater when Pro is in the interruption, GP, than when hydrophobic residues (GF, GY) are present, and a rigid Gly-Pro-Hyp tripeptide adjacent to the interruption leads to greater destabilization than a flexible Gly-Ala-Ala sequence. Modeling based on NMR data indicates the Phe residue within a GF interruption is located on the outside of the triple helix. The G1G interruptions resemble a previously studied collagen interruption GPOGAAVMGPO, designated G4G-type, in that both are destabilizing, but allow continuation of rod-like triple helices and maintenance of the single residue stagger throughout the imperfection, with a loss of axial register of the superhelix on both sides. Both kinds of interruptions result in a highly localized perturbation in hydrogen bonding and dihedral angles, but the hydrophobic residue of a G4G interruption packs near the central axis of the superhelix, while the hydrophobic residue of a G1G interruption is located on the triple-helix surface. The different structural consequences of G1G and G4G interruptions in the repeating tripeptide sequence pattern suggest a physical basis for their differential susceptibility to matrix metalloproteinases in type X collagen.     

Co-translational incorporation of trans-4-hydroxyproline into recombinant proteins in bacteria

Trans-4-hydroxyproline (Hyp) in eukaryotic proteins arises from post-translational modification of proline residues. Because the modification enzyme is not present in prokaryotes, no natural means exists to incorporate Hyp into proteins synthesized in Escherichia coli. We show here that under appropriate culture conditions Hyp is incorporated co-translationally directly at proline codons in genes expressed in E. coli. The use of Hyp by E. coli protein synthesis machinery under typical culture conditions is not adequate to support protein synthesis; however, intracellular concentrations of Hyp sufficient to compensate for the poor use are achieved in media with hyperosmotic sodium chloride concentrations. Hyp incorporation was demonstrated in several recombinant proteins including human Type I collagen polypeptides. A fragment of the human collagen Type I (alpha1) polypeptide with global Hyp for Pro substitution forms a triple helix. Our results demonstrate a remarkable pliancy in the biosynthetic apparatus of bacteria that may be used more generally to incorporate novel amino acids into recombinant proteins.     

The NC2 Domain of Collagen IX Provides Chain Selection and Heterotrimerization

The mechanism of chain selection and trimerization of fibril-associated collagens with interrupted triple helices (FACITs) differs from that of fibrillar collagens that have special C-propeptides. We recently showed that the second carboxyl-terminal non-collagenous domain (NC2) of homotrimeric collagen XIX forms a stable trimer and substantially stabilizes a collagen triple helix attached to either end. We then hypothesized a general trimerizing role for the NC2 domain in other FACITs. Here we analyzed the NC2 domain of human heterotrimeric collagen IX, the only member of FACITs with all three chains encoded by distinct genes. Upon oxidative folding of equimolar amounts of the α1, α2, and α3 chains of NC2, a stable heterotrimer with a disulfide bridge between α1 and α3 chains is formed. Our experiments show that this heterotrimerization domain can stabilize a short triple helix attached at the carboxyl-terminal end and allows for the proper oxidation of the cystine knot of type III collagen after the short triple helix.     

Electrostatic interactions involving lysine make major contributions to collagen triple-helix stability

Important stabilizing features for the collagen triple helix include the presence of Gly as every third residue, a high content of imino acids, and interchain hydrogen bonds. Host-guest peptides have been used previously to characterize triple-helix propensities of individual residues and Gly-X-Y triplets. Here, comparison of the thermal stabilities of host-guest peptides of the form (Gly-Pro-Hyp)3-Gly-X-Y-Gly-X'-Y'-(Gly-Pro-Hyp)3 extends the study to adjacent tripeptide sequences, to encompass the major classes of potential direct intramolecular interactions. Favorable hydrophobic interactions were observed, as well as stabilizing intrachain interactions between residues of opposite charge in the i and i + 3 positions. However, the greatest gain in triple-helix stability was achieved in the presence of Gly-Pro-Lys-Gly-Asp/Glu-Hyp sequences, leading to a T(m) value equal to that seen for a Gly-Pro-Hyp-Gly-Pro-Hyp sequence. This stabilization is seen for Lys but not for Arg and can be assigned to interchain ion pairs, as shown by molecular modeling. Computational analysis shows that Lys-Gly-Asp/Glu sequences are present at a frequency much greater than expected in collagen, suggesting this interaction is biologically important. These results add significantly to the understanding of which surface ion pairs can contribute to protein stability.     

Crystal structure of human type III collagen Gly991-Gly1032 cystine knot-containing peptide shows both 7/2 and 10/3 triple helical symmetries

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.     

Vascular Ehlers-Danlos Syndrome Mutations in Type III Collagen Differently Stall the Triple Helical Folding

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.     

Type XIV collagen, a new homotrimeric molecule extracted from fetal bovine skin and tendon, with a triple helical disulfide-bonded domain homologous to type IX and type XII collagens

Previously undescribed disulfide-bonded collagenous pepsin-derived fragments have been isolated from fetal calf tendon and skin. One fragment, 10.5 kDa after reduction, was shown to be similar but distinct to the COL1 domain of the recently characterized type XII collagen (64% primary structure identity). The similarity includes important features such as size, location of the cysteine residues, and nature and position of an imperfection of the triple helix. From fetal calf skin, two approximately 34-kDa disulfide-bonded trimeric fragments were isolated in the unreduced form. Amino acid sequencing showed that one fragment contained solely the COL1 domain of type XII collagen while the other one only contained the COL1 domain of the new chain. Like type XII collagen, the new chain is therefore part of a homotrimeric molecule and should thus be considered as a distinct collagen type. We propose to call the molecule from which this fragment is derived, type XIV collagen, with a chain composition (alpha 1 (XIV]3. The presence of a domain similar to the COL1 domain of collagens types IX and XII suggests that type XIV collagen belongs to the group of fibril-associated collagens with interrupted triple helices (FACIT). Two other fragments, 13.5 and 17 kDa after reduction, were also purified. They were shown to contain the same triple helical domain with different pepsin cleavage sites at the amino terminus. Several tryptic peptides were sequenced, and the derived sequences could be aligned with the COL2 domain of type XII collagen or with flanking sequences in the NC2 and NC3 domains (61% sequence identity). These fragments are very likely to be also derived from type XIV collagen.     

Recombinant collagen studies link the severe conformational changes induced by osteogenesis imperfecta mutations to the disruption of a set of interchain salt bridges

The clinical severity of Osteogenesis Imperfecta (OI), also known as the brittle bone disease, relates to the extent of conformational changes in the collagen triple helix induced by Gly substitution mutations. The lingering question is why Gly substitutions at different locations of collagen cause different disruptions of the triple helix. Here, we describe markedly different conformational changes of the triple helix induced by two Gly substitution mutations placed only 12 residues apart. The effects of the Gly substitutions were characterized using a recombinant collagen fragment modeling the 63-residue segment of the alpha1 chain of type I collagen containing no Hyp (residues 877-939) obtained from Escherichia coli. Two Gly --> Ser substitutions at Gly-901 and Gly-913 associated with, respectively, mild and severe OI variants were introduced by site-directed mutagenesis. Biophysical characterization and limited protease digestion experiments revealed that while the substitution at Gly-901 causes relatively minor destabilization of the triple helix, the substitution at Gly-913 induces large scale unfolding of an unstable region C-terminal to the mutation site. This extensive unfolding is caused by the intrinsic low stability of the C-terminal region of the helix and the mutation induced disruption of a set of salt bridges, which functions to lock this unstable region into the triple helical conformation. The extensive conformational changes associated with the loss of the salt bridges highlight the long range impact of the local interactions of triple helix and suggest a new mechanism by which OI mutations cause severe conformational damages in collagen.     

Genome-based identification and analysis of collagen-related structural motifs in bacterial and viral proteins

Collagens are extended trimeric proteins composed of the repetitive sequence glycine-X-Y. A collagen-related structural motif (CSM) containing glycine-X-Y repeats is also found in numerous proteins often referred to as collagen-like proteins. Little is known about CSMs in bacteria and viruses, but the occurrence of such motifs has recently been demonstrated. Moreover, bacterial CSMs form collagen-like trimers, even though these organisms cannot synthesize hydroxyproline, a critical residue for the stability of the collagen triple helix. Here we present 100 novel proteins of bacteria and viruses (including bacteriophages) containing CSMs identified by in silico analyses of genomic sequences. These CSMs differ significantly from human collagens in amino acid content and distribution; bacterial and viral CSMs have a lower proline content and a preference for proline in the X position of GXY triplets. Moreover, the CSMs identified contained more threonine than collagens, and in 17 of 53 bacterial CSMs threonine was the dominating amino acid in the Y position. Molecular modeling suggests that threonines in the Y position make direct hydrogen bonds to neighboring backbone carbonyls and thus substitute for hydroxyproline in the stabilization of the collagen-like triple-helix of bacterial CSMs. The majority of the remaining CSMs were either rich in proline or rich in charged residues. The bacterial proteins containing a CSM that could be functionally annotated were either surface structures or spore components, whereas the viral proteins generally could be annotated as structural components of the viral particle. The limited occurrence of CSMs in eubacteria and lower eukaryotes and the absence of CSMs in archaebacteria suggests that DNA encoding CSMs has been transferred horizontally, possibly from multicellular organisms to bacteria.     

Dissecting a bacterial collagen domain from Streptococcus pyogenes: sequence and length-dependent variations in triple helix stability and folding

To better investigate the relationship between sequence, stability, and folding, the Streptococcus pyogenes collagenous domain CL (Gly-Xaa-Yaa)(79) was divided to create three recombinant triple helix subdomains A, B, and C of almost equal size with distinctive amino acid features: an A domain high in polar residues, a B domain containing the highest concentration of Pro residues, and a very highly charged C domain. Each segment was expressed as a monomer, a linear dimer, and a linear trimer fused with the trimerization domain (V domain) in Escherichia coli. All recombinant proteins studied formed stable triple helical structures, but the stability varied depending on the amino acid sequence in the A, B, and C segments and increased as the triple helix got longer. V-AAA was found to melt at a much lower temperature (31.0 °C) than V-ABC (V-CL), whereas V-BBB melted at almost the same temperature (～36-37 °C). When heat-denatured, the V domain enhanced refolding for all of the constructs; however, the folding rate was affected by their amino acid sequences and became reduced for longer constructs. The folding rates of all the other constructs were lower than that of the natural V-ABC protein. Amino acid substitution mutations at all Pro residues in the C fragment dramatically decreased stability but increased the folding rate. These results indicate that the thermostability of the bacterial collagen is dominated by the most stable domain in the same manner as found with eukaryotic collagens.     

The collagen-like peptide (GER)15GPCCG forms pH-dependent covalently linked triple helical trimers

A collagen-like peptide with the sequence (GER)(15) GPCCG was synthesized to study the formation of a triple helix in the absence of proline residues. This peptide can form a triple helix at acidic and basic pH, but is insoluble around neutral pH. The formation of a triple helix can be used to covalently oxidize the cysteine residues into a disulfide knot. Three disulfide bonds are formed between the three chains as has been found at the carboxyl-terminal end of the type III collagen triple helix. This is a new method to covalently link collagen-like peptides with a stereochemistry that occurs in nature. The peptide undergoes a reversible, cooperative triple helix coil transition with a transition midpoint (T(m)) of 17 to 20 degrees C at acidic pH and 32 to 37 degrees C at basic pH. At acidic pH there was little influence of the T(m) on the salt concentration of the buffer. At basic pH increasing the salt concentration reduced the T(m) to values comparable to the stability at acidic pH. These experiments show that the tripeptide unit GER which occurs frequently in collagen sequences can form a triple helical structure in the absence of more typical collagen-like tripeptide units and that charge-charge interactions play a role in the stabilization of the triple helix of this peptide.     

Inter-chain Proline: Proline Contacts Contribute to the Stability of the Triple Helical Conformation

Abstract

The triple helical conformation observed in the collagen group of proteins is related to the presence of large numbers of imino residues and is derived from the stereochemical properties of these residues. The triple helix is stabilized by increasing numbers of these residues. Hydrogen bonds are usually considered to be a major factor in the formation and stability of protein conformation, however, imino residues are not hydrogen bond donors. We have evaluated the role of these residues in stabilizing the triple helix by re-examining two X-ray based structures of the triple helical polypeptide (Pro-Pro- Gly)₁₀ using molecular mechanics calculations. The two minimized structures are comparable in energy and have helical parameters close to the starting values for each starting structure. Our studies suggest that clusters of close van der Waals contacts between proline residues in adjacent chains contribute significantly to the stability of the triple helix. Preliminary NMR studies support this concept. We propose that non-bonded interactions between proline residues may be a significant stabilizing force in the triple helix generated by (Pro-Pro-Gly)₁₀.