Interruptions in the collagen repeating tripeptide pattern can promote supramolecular association

The standard collagen triple‐helix requires a perfect (Gly‐Xaa‐Yaa)_n sequence, yet all nonfibrillar collagens contain interruptions in this tripeptide repeating pattern. Defining the structural consequences of disruptions in the sequence pattern may shed light on the biological role of sequence interruptions, which have been suggested to play a role in molecular flexibility, collagen degradation, and ligand binding. Previous studies on model peptides with 1‐ and 4‐residue interruptions showed a localized perturbation within the triple‐helix, and this work is extended to introduce natural collagen interruptions up to nine residue in length within a fixed (Gly‐Pro‐Hyp)_n peptide context. All peptides in this set show decreases in triple‐helix content and stability, with greater conformational perturbations for the interruptions longer than five residue. The most stable and least perturbed structure is seen for the 5‐residue interruption peptide, whose sequence corresponds to a Gly to Ala missense mutation, such as those leading to collagen genetic diseases. The triple‐helix peptides containing 8‐ and 9‐residue interruptions exhibit a strong propensity for self‐association to fibrous structures. In addition, a small peptide modeling only the 9‐residue sequence within the interruption aggregates to form amyloid‐like fibrils with antiparallel β‐sheet structure. The 8‐ and 9‐residue interruption sequences studied here are predicted to have significant cross‐β aggregation potential, and a similar propensity is reported for ～10% of other naturally occurring interruptions. The presence of amyloidogenic sequences within or between triple‐helix domains may play a role in molecular association to normal tissue structures and could participate in observed interactions between collagen and amyloid.     

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.     

Triple-helical peptides: an approach to collagen conformation, stability, and self-association

Peptides have been an integral part of the collagen triple-helix structure story, and have continued to serve as useful models for biophysical studies and for establishing biologically important sequence-structure-function relationships. High resolution structures of triple-helical peptides have confirmed the basic Ramachandran triple-helix model and provided new insights into the hydration, hydrogen bonding, and sequence dependent helical parameters in collagen. The dependence of collagen triple-helix stability on the residues in its (Gly-X-Y)(n) repeating sequence has been investigated by measuring melting temperatures of host-guest peptides and an on-line collagen stability calculator is now available. Although the presence of Gly as every third residue is essential for an undistorted structure, interruptions in the repeating (Gly-X-Y)(n) amino acid sequence pattern are found in the triple-helical domains of all nonfibrillar collagens, and are likely to play a role in collagen binding and degradation. Peptide models indicate that small interruptions can be incorporated into a rod-like triple-helix with a highly localized effect, which perturbs hydrogen bonds and places the standard triple-helices on both ends out of register. In contrast to natural interruptions, missense mutations which replace one Gly in a triple-helix domain by a larger residue have pathological consequences, and studies on peptides containing such Gly substitutions clarify their effect on conformation, stability, and folding. Recent studies suggest peptides may also be useful in defining the basic principles of collagen self-association to the supramolecular structures found in tissues.     

Molecular dynamic simulation studies on the effect of one residue chain staggering on the structure and stability of heterotrimeric collagen-like peptides with interruption

A systematic molecular dynamics (MD) simulation has been carried out on collagen-like peptides with different combinations of interruptions in the Gly-X(AA) -Y(AA) repeats. Although experimental studies have been carried out to elucidate the structural consequences of homotrimeric collagen-like peptides, this is the first report on the structural effect on the heterotrimeric models with G4G and G1G breaks present simultaneously in the constituent chains with difference in one residue chain staggering. The results reveal that the axial registry of the interrupted region changes significantly from that of conventional triple helical peptide without interruption. Further, results from MD simulations show the formation of a kink in the interrupted region of the triple-helical peptides. The conformational analysis reveals that the interruption in the Gly-X(AA) -Y(AA) pattern in these peptides induces β-strand conformation in triple helical peptides. The conventional hydrogen bonds in the interrupted triad are affected and new nonconventional H-bonds are formed in the triple helical structure, and as a result interrupted region becomes locally fragile. MM-PBSA calculations on the different systems clearly suggest that the binding affinity varies marginally due to one residue staggering. However, it is found from the structural parameters that hydrogen-bonding pattern differs significantly due to the difference in the staggering of chains.     

Atypical gly-X-Y sequences surround interruptions in the repeating tripeptide pattern of basement membrane collagen

The triple-helical domains of type IV collagen chains have more than 20 sites at which the repeating (Gly-X-Y)_n pattern is interrupted. Analysis of α1 (IV) and α2 (IV) chains indicates the residues in the three Gly-X-Y triplets preceding or following interruptions differ statistically from the rest of the chain. Unusually high frequencies of charged residues are seen at a number of X and Y sites, with the charge density being particularly high C-terminal to the interruption site. Analyses were carried out on individual categories of interruptions, classified as insertions or deletions in the Y position. All of the residues in the X and Y positions of the triplets flanking insertion sites are atypical, with a high concentration of charged residues. Triplets flanking sites where there has been a deletion in the Y position show unusually high frequencies of charged residues at some sites, hydrophobic residues at other sites, and an invariant imino acid N-terminal to the interruption. The presence of atypical sequences surrounding interruptions could be important at a molecular level, related to triple-helix stability, or at a supramolecular level, related to the association of molecules to form networks in basement membranes. © 1995 John Wiley & Sons, Inc.     

Conformational effects of Gly-X-Gly interruptions in the collagen triple helix

The collagen model peptide with sequence (Pro-Hyp-Gly)4-Pro-Gly-(Pro-Hyp-Gly)5 contains a central Gly-Pro-Gly interruption in the consensus collagen sequence. Its high-resolution crystal structure defines the molecular consequences of such an interruption for the collagen triple-helical conformation, and provides insight into possible structural and biological roles of similar interruptions in the -Gly-X-Y- repeating pattern found in non-fibrillar collagens. The peptide (denoted as the Hyp minus peptide or Hyp-) forms a rod-like triple helix structure without any bend or kink, and crystallizes in a quasi-hexagonal lattice. The two Pro-Hyp-Gly zones adopt the typical triple-helical collagen conformation with standard Rich and Crick II hydrogen bonding topology. Notably, the central zone containing the Gly-Pro-Gly interruption deviates from the standard structure in terms of hydrogen bonding topology, torsion angles, helical, and superhelical parameters. These deviations are highly localized, such that the standard features are regained within one to two residues on either side. Conformational variations and high temperature factors seen for the six chains of the asymmetric unit in the zone around the interruption point to the presence of a local region of considerable plasticity and flexibility embedded within two highly rigid and ordered standard triple-helical segments. The structure suggests a role for Gly-X-Gly interruptions as defining regions of flexibility and molecular recognition in the otherwise relatively uniform repeating collagen conformation.     

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.     

Transformation of the mechanism of triple-helix peptide folding in the absence of a C-terminal nucleation domain and its implications for mutations in collagen disorders

Folding abnormalities of the triple helix have been demonstrated in collagen diseases such as osteogenesis imperfecta in which the mutation leads to the substitution of a single Gly in the (Gly-X-Y)n sequence pattern by a larger residue. Model peptides can be used to clarify the details of normal collagen folding and the consequences of the interruption of that folding by a Gly substitution. NMR and CD studies show that placement of a (GPO)4 nucleation domain at the N terminus rather than the C terminus of a native collagen sequence allows the formation of a stable triple helix but alters the folding mechanism. Although C- to N-terminal directional folding occurs when the nucleation domain is at the C terminus, there is no preferential folding direction when the nucleation domain is at the N terminus. The lack of zipper-like directional folding does not interfere with triple-helix formation, and when a Gly residue is replaced by Ser to model an osteogenesis imperfecta mutation, the peptide with the N-terminal (GPO)4 domain can still form a good triple helix N-terminal to the mutation site. These peptide studies raise the possibility that mutant collagen could fold in a C to N direction in a zipper-like manner up to the mutation site and that completion of the triple helix N-terminal to the mutation would involve an alternative mechanism.     

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.     

Quantum-mechanical study of model molecules in relation to collagen structure. II. (Gly-Phe-Pro) and (Gly-Pro-Phe) repeating units

A conformational quantum-mechanical study of (Gly-Phe-Pro) and (Gly-Pro-Phe) repeating tripeptide sequences has been carried out with the PCILO method. Using appropriate molecules as a model, we investigated the conformational possibilities of each in situ residue. Computations have been done taking into account the two typical pyrrolidine ring puckering and the most favorable orientations of the phenylalanyl side chain. Major conclusions drawn from this study are that the phenylalanyl can be accommodated at both second and third positions in the sequence without preventing the formation of triple-helix conformation. However, the analogy observed between the rotational possibilities around the second residue of Gly-Pro-Pro and Gly-Phe-Pro indicates that phenylalanyl in the second position favors the triple-helix formation.     

Sequence specific thermal stability of the collagen triple helix

Theoretical calculations of the thermal stability of collagen triple helices using empirical values for the contribution of individual tripeptide units are presented and compared with direct measurements of the thermal stability of various types of collagens. Relative stabilities are assigned to the positions of the tripeptide units in the amino acid sequence along the length of the collagen molecule. The sequence specific relative stabilities of type I and type XI collagens are compared. These offer insight into the reasons for the existence of unfolding intermediates in type XI collagen that are absent in type I collagen. The pattern of relative stabilities calculated for mouse type IV collagen is consistent with experimental results which indicate that the amino terminal region is very stable and that the interruptions cause increased flexibility and independently unfolding domains. Mutations in the triple helical domain of human type I procollagen occurring in brittle bone disease (osteogenesis imperfecta) show varying effects on the thermal stability of the molecule. The sequence specific thermal stability calculations shed some light on why some mutations of cysteine for glycine have greater effects on the thermal stability than others.     

NMR shows hydrophobic interactions replace glycine packing in the triple helix at a natural break in the (Gly-X-Y)n repeat

Little is known about the structural consequences of the more than 20 breaks in the (Gly-X-Y)(n) repeating sequence found in the long triple helix domain of basement membrane type IV collagen. NMR triple resonance studies of doubly labeled residues within a set of collagen model peptides provide distance and dihedral angle restraints that allow determination of model structures of both a standard triple helix and of a triple helix with a break in solution. Although the standard triple helix cannot continue when Gly is not every third residue, the NMR data support rod-like molecules that have standard triple-helical structures on both sides of a well defined and highly localized perturbation. The GAAVM break region may be described as a "pseudo triple helix," because it preserves the standard one-residue stagger of the triple helix but introduces hydrophobic interactions at the position normally occupied by the much smaller and hydrogen-bonded Gly residue of the repeating (Gly-X-Y)(n) sequence. This structure provides a rationale for the consensus presence of hydrophobic residues in breaks of similar length and defines a novel variant of a triple helix that could be involved in recognition.     

The Self-assembly of a Mini-fibril with Axial Periodicity from a Designed Collagen-mimetic Triple Helix

In this work we describe the self-assembly of a collagen-like periodic mini-fibril from a recombinant triple helix. The triple helix, designated Col108, is expressed in Escherichia coli using an artificial gene and consists of a 378-residue triple helix domain organized into three pseudo-repeating sequence units. The peptide forms a stable triple helix with a melting temperature of 41 °C. Upon increases of pH and temperature, Col108 self-assembles in solution into smooth mini-fibrils with the cross-striated banding pattern typical of fibrillar collagens. The banding pattern is characterized by an axially repeating feature of ∼35 nm as observed by transmission electron microscopy and atomic force microscopy. Both the negatively stained and the positively stained transmission electron microscopy patterns of the Col108 mini-fibrils are consistent with a staggered arrangement of triple helices having a staggering value of 123 residues, a value closely connected to the size of one repeat sequence unit. A mechanism is proposed for the mini-fibril formation of Col108 in which the axial periodicity is instigated by the built-in sequence periodicity and stabilized by the optimized interactions between the triple helices in a 1-unit staggered arrangement. Lacking hydroxyproline residues and telopeptides, two factors implicated in the fibrillogenesis of native collagen, the Col108 mini-fibrils demonstrate that sequence features of the triple helical domain alone are sufficient to “code” for axially repeating periodicity of fibrils. To our knowledge, Col108 is the first designed triple helix to self-assemble into periodic fibrils and offers a unique opportunity to unravel the specific molecular interactions of collagen fibrillogenesis.     

Sequence dependence of renucleation after a Gly mutation in model collagen peptides

Missense mutations in the collagen triple helix that replace one Gly residue in the (Gly-X-Y)(n) repeating pattern by a larger amino acid have been shown to delay triple helix folding. One hypothesis is that such mutations interfere with the C- to N-terminal directional propagation and that the identity of the residues immediately N-terminal to the mutation site may determine the delay time and the degree of clinical severity. Model peptides are designed to clarify the role of tripeptide sequences N-terminal to the mutation site, with respect to length, stability, and nucleation propensity, to complete triple helix folding. Two sets of peptides with different N-terminal sequences, one with the natural sequence alpha1(I) 886-900, which is just adjacent to the Gly(901) mutation, and one with a GPO(GAO)(3) sequence, which occurs at alpha1(I) 865-879, are studied by CD and NMR. Placement of the five tripeptides of the natural alpha1(I) collagen sequence N-terminal to the Gly to Ala mutation site results in a peptide that is folded only C-terminal to the mutation site. In contrast, the presence of the Hyp-rich sequence GPO(GAO)(3) N-terminal to the mutation allows complete refolding in the presence of the mutation. The completely folded peptide contains an ordered central region with unusual hydrogen bonding while maintaining standard triple helix structure at the N- and C-terminal ends. These peptide results suggest that the location and sequences of downstream regions favorable for renucleation could be the key factor in the completion of a triple helix N-terminal to a mutation.     

Conformational features of a natural break in the type IV collagen Gly-X-Y repeat

Fibrillar collagens have an absolute requirement for Gly as every 3rd residue, whereas breaks in the Gly-X-Y repeating pattern are found normally in the triple helix domains of non-fibrillar collagens, such as type IV collagen in basement membranes. In this study, a model 30-mer peptide is designed to include the interruption GPOGAAVMGPOGPO found in the alpha5 chain of type IV collagen. The GAAVM peptide forms a stable triple helix, with Tm= 29 degrees C. When compared with a control peptide with Gly as every 3rd residue, the GAAVM peptide has a marked decrease in the 225 nm maximum of its CD spectrum and a 10 degrees C drop in stability. A 50% decrease in calorimetric enthalpy is observed, which may result from disruption of ordered water structure anchored by regularly placed backbone carbonyls. NMR studies on specific 15N-labeled residues within the GAAVM peptide indicate a normal triple helical structure for Gly-Pro-Hyp residues flanking the break. The sequence within the break is not disordered but shows altered hydrogen exchange rates and an abnormal Val chemical shift. It was previously reported that a peptide designed to model a similar kind of interruption in the peptide (Pro-Hyp-Gly)10, (GPOGPOPOGPO), is unable to form a stable triple helix, and replacement of GAA by GPO or VM by PO within the GAAVM break decreases the stability. Thus, rigid imino acids are unfavorable within a break, despite their favorable stabilization of the triple helix itself. These results suggest some non-random structure typical of this category of breaks in the Gly-X-Y repeat of the triple helix.     

Structural model of the collagen-like region of C1q comprising the kink region and the fibre-like packing of the six triple helices

A detailed three-dimensional model of the collagenous part of C1q was derived by model building and computer-aided energy refinement calculations. The proposed structure is based on the collagen-like (-Gly-Xaa-Yaa-) repeating sequence of 78 to 81 residues in the N-terminal regions of the constituent A, B and C chains, on the mode of disulphide linkage between the 18 chains of C1q, and on its electron microscopically derived gross structure. It is demonstrated that the interruptions of the repeating sequence about half-way along the length of the collagenous regions (Gly36-Ile37-Arg38-Thr39 in the A chain and Ala36-Ile37-Hy138 in the C chain) do not lead to a disruption of the triple helical conformation but rather to a bend of about 60 degrees in an otherwise continuous triple helix. These features are consistent with a flexibility comparable with that of regular triple helices and with the observed low proteolytic susceptibility of the kink region. The azimuthal orientation of the kink is defined approximately by ArgA38 being located in the cap of the knee. Because of this extra residue between two glycine residues, a bad contact that would arise between the methyl group of AlaC36 and the peptide carbonyl of IleA37 in a straight triple helix is relaxed. The model features also a cluster of hydrophobic contacts between large hydrophobic side-chains in the interaction edges between the six collagen triple helices aligned with their about 10 nm long N-terminal regions in the fibril-like endpiece of C1q. The azimuthal orientations of the triple helices were derived by energy calculations of side-chain interactions previously applied to fibre-forming collagens. Independently, the same orientations and interaction edges were derived from the azimuthal orientation of the kink and the electron microscopically observed orientations of the triple helical arms that emerge from the endpiece, and which carry the C-terminal globular binding domains. The structural model has a number of implications for the assembly of the first component of complement from C1q and the zymogen complex C1r2C1s2 and possible mechanisms of its activation.     

Osteogenesis imperfecta missense mutations in collagen: structural consequences of a glycine to alanine replacement at a highly charged site

Glycine is required as every third residue in the collagen triple helix, and a missense mutation leading to the replacement of even one Gly in the repeating (Gly-Xaa-Yaa)(n) sequence with a larger residue leads to a pathological condition. Gly to Ala missense mutations are highly underrepresented in osteogenesis imperfecta (OI) and other collagen diseases, suggesting that the smallest replacement residue, Ala, might cause the least structural perturbation and mildest clinical consequences. The relatively small number of Gly to Ala mutation sites that do lead to OI must have some unusual features, such as greater structural disruption because of local sequence environment or location at a biologically important site. Here, peptides are used to model a severe OI case in which a Gly to Ala mutation is found within a highly stabilizing Lys-Gly-Asp sequence environment. Nuclear magnetic resonance, circular dichroism, and differential scanning calorimetry studies indicate this Gly to Ala replacement leads to a substantial loss of triple-helix stability and nonequivalence of the Ala residues in the three chains such that only one of the three Ala residues is capable of forming a good backbone hydrogen bond. Examination of reported OI Gly to Ala mutations suggests their preferential location at known collagen binding sites, and we propose that structural defects caused by Ala replacements may lead to pathology when they interfere with interactions.     

A statistically derived parameterization for the collagen triple-helix

The triple-helix is a unique secondary structural motif found primarily within the collagens. In collagen, it is a homo- or hetero-tripeptide with a repeating primary sequence of (Gly-X-Y)(n), displaying characteristic peptide backbone dihedral angles. Studies of bulk collagen fibrils indicate that the triple-helix must be a highly repetitive secondary structure, with very specific constraints. Primary sequence analysis shows that most collagen molecules are primarily triple-helical; however, no high-resolution structure of any entire protein is yet available. Given the drastic morphological differences in self-assembled collagen structures with subtle changes in assembly conditions, a detailed knowledge of the relative locations of charged and sterically bulky residues in collagen is desirable. Its repetitive primary sequence and highly conserved secondary structure make collagen, and the triple-helix in general, an ideal candidate for a general parameterization for prediction of residue locations and for the use of a helical wheel in the prediction of residue orientation. Herein, a statistical analysis of the currently available high-resolution X-ray crystal structures of model triple-helical peptides is performed to produce an experimentally based parameter set for predicting peptide backbone and C(beta) atom locations for the triple-helix. Unlike existing homology models, this allows easy prediction of an entire triple-helix structure based on all existing high-resolution triple-helix structures, rather than only on a single structure or on idealized parameters. Furthermore, regional differences based on the helical propensity of residues may be readily incorporated. The parameter set is validated in terms of the predicted bond lengths, backbone dihedral angles, and interchain hydrogen bonding.     

Proposed alignment of helical interruptions in the two subunits of the basement membrane (type IV) collagen

We have isolated a cDNA clone for part of the alpha 2 type IV collagen (pCIV-2-176). Deoxynucleotide sequence analysis shows that this clone codes for 439 amino acids from the helical domain adjacent to the C-terminal globular domain of the alpha 2 (IV) chain. By aligning the deduced amino acid sequence of the alpha 2 (IV) chain with the published sequence for the alpha 1 (IV) chain, we find that all interruptions in the alpha 1 (IV) chain coincide with an interruption in the alpha 2 (IV) chain. Additional interruptions in the alpha 2 (IV) chain exist, however, three out of the four analysed only slightly disturb the collagen triple helix.     

Location of glycine mutations within a bacterial collagen protein affects degree of disruption of triple-helix folding and conformation

The hereditary bone disorder osteogenesis imperfecta is often caused by missense mutations in type I collagen that change one Gly residue to a larger residue and that break the typical (Gly-Xaa-Yaa)(n) sequence pattern. Site-directed mutagenesis in a recombinant bacterial collagen system was used to explore the effects of the Gly mutation position and of the identity of the residue replacing Gly in a homogeneous collagen molecular population. Homotrimeric bacterial collagen proteins with a Gly-to-Arg or Gly-to-Ser replacement formed stable triple-helix molecules with a reproducible 2 °C decrease in stability. All Gly replacements led to a significant delay in triple-helix folding, but a more dramatic delay was observed when the mutation was located near the N terminus of the triple-helix domain. This highly disruptive mutation, close to the globular N-terminal trimerization domain where folding is initiated, is likely to interfere with triple-helix nucleation. A positional effect of mutations was also suggested by trypsin sensitivity for a Gly-to-Arg replacement close to the triple-helix N terminus but not for the same replacement near the center of the molecule. The significant impact of the location of a mutation on triple-helix folding and conformation could relate to the severe consequences of mutations located near the C terminus of type I and type III collagens, where trimerization occurs and triple-helix folding is initiated.