The Streptococcus pyogenes cell-surface protein Scl2 contains a globular N-terminal domain and a collagen-like domain, (GlyXaa-Xaa) 79 , which forms a triple helix with a thermal stability close to that seen for mammalian collagens. Hyp is a major contributor to triple-helix stability in animal collagens, but is not present in bacteria, which lack prolyl hydroxylase. To explore the basis of bacterial collagen triple-helix stability in the absence of Hyp, biophysical studies were carried out on recombinant Scl2 protein, the isolated collagen-like domain from Scl2, and a set of peptides modeling the Scl2 highly charged repetitive (GlyXaa-Xaa) n sequences. At pH 7, CD spectroscopy, dynamic light scattering, and differential scanning calorimetry of the Scl2 protein all showed a very sharp thermal transition near 36°C, indicating a highly cooperative unfolding of both the globular and triple-helix domains. The collagen-like domain isolated by trypsin digestion showed a sharp transition at the same temperature, with an enthalpy of 12.5 kJ/mol of tripeptide. At low pH, Scl2 and its isolated collagen-like domain showed substantial destabilization from the neutral pH value, with two thermal transitions at 24 and 27°C. A similar destabilization at low pH was seen for Scl2 charged model peptides, and the degree of destabilization was consistent with the strong pH dependence arising from the GKD tripeptide unit. The Scl2 protein contained twice as much charge as human fibril-forming collagens, and the degree of electrostatic stabilization observed for Scl2 was similar to the contribution Hyp makes to the stability of mammalian collagens. The high enthalpic contribution to the stability of the Scl2 collagenous domain supports the presence of a hydration network in the absence of Hyp.Collagens are considered to be the characteristic structural molecules of the extracellular matrix of multicellular animals. Fibril-forming collagens and basement membrane collagens are ubiquitous in vertebrates and invertebrates, whereas families of more specialized collagens have developed in different organisms such as the 28 distinct collagen types found in vertebrates (1-3) and the ϳ100 cuticle collagen genes in Caenorhabditis elegans (4). In recent years, the range of occurrence of collagen-like sequences with Gly as every 3rd residue and a high Pro content has been extended from metazoans to Ͼ100 proteins in bacteria and bacteriophage (5). An understanding of the structure and stabilization of such bacterial collagens presents new challenges because they lack the Hyp post-translational modification characteristic of animal collagens.A high content of Hyp is a unique stabilizing feature of animal collagens. The characteristic structural motif of all collagens is the triple helix, composed of three left-handed polyproline II-type chains (3 residues/turn) wound around the central axis to form a right-handed superhelix (6 -8). The close packing of each chain near the central axis constrains every 3rd residue of the amino acid sequence to be G...

Interest in self-association of peptides and proteins is motivated by an interest in the mechanism of physiologically higher order assembly of proteins such as collagen as well as the mechanism of pathological aggregation such as ␤-amyloid formation. The triple helical form of (Pro-Hyp-Gly) 10 , a peptide that has proved a useful model for molecular features of collagen, was found to self-associate, and its association properties are reported here. Turbidity experiments indicate that the triple helical peptide self-assembles at neutral pH via a nucleationgrowth mechanism, with a critical concentration near 1 mM. The associated form is more stable than individual molecules by about 25°C, and the association is reversible. The rate of self-association increases with temperature, supporting an entropically favored process. After self-association, (ProHyp-Gly) 10 forms branched filamentous structures, in contrast with the highly ordered axially periodic structure of collagen fibrils. Yet a number of characteristics of triple helix assembly for the peptide resemble those of collagen fibril formation. These include promotion of fibril formation by neutral pH and increasing temperature; inhibition by sugars; and a requirement for hydroxyproline. It is suggested that these similar features for peptide and collagen self-association are based on common lateral underlying interactions between triple helical molecules mediated by hydrogen-bonded hydration networks involving hydroxyproline.There is increasing interest in the ability of proteins and peptides to self-associate into aggregates, both in normal and pathological processes. Normal self-association processes include fibril formation of collagen and polymerization of actin (1, 2), whereas pathological aggregation of amyloid peptides, ␣-synuclein, and prions is implicated in neurodegenerative diseases (3,4). Interest has focused on the nature of protein aggregation and the molecular and environmental determinants of the self-association process. The study of the ability of collagen-like peptides to aggregate offers an opportunity to characterize a unique system, which may relate to the physiological self-association of collagen molecules.Collagen, the major structural protein in the extracellular matrix, has a characteristic triple helical conformation, consisting of three polyproline II-like chains that are supercoiled around a common axis (5-7). The close packing of the three chains near the central axis generates a requirement for Gly as every third residue, (Gly-X-Y) n , whereas the high content of imino acids Pro and hydroxyproline (Hyp) stabilizes the individual polyproline II-like helices. Although imino acids are highly favorable for the triple helix, the post-translational modification of Pro to Hyp in the Y position confers an additional stabilizing contribution. This further stabilization of Hyp is likely to result from steroelectronic promotion of the more favorable exo ring pucker for the Y position and Hyp involvement in solvent-mediated hydrogen bonding...

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 report defines the sequence features and structural consequences of the most common interruption, where one residue is missing in the tripeptide pattern, Gly-X1-X2-Gly-AA 1 -Gly-X1-X2, designated as 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 a 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 as a G4G type, in that both are destabilizing, but allow continuation of rod-like triple-helices and maintenance of the 1-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.

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 ␣5 chain of type IV collagen. The GAAVM peptide forms a stable triple helix, with T m ‫؍‬ 29°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°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 15 N-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 , (GPOG-POPOGPO), 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.The repeating (Gly-X-Y) n sequence of the collagen triple helix allows for easy identification of this structural motif from amino acid sequences. The collagen triple helix structure consists of three supercoiled polypeptide chains, each in a polyproline II-like conformation (1-4). The repeating pattern of Gly as every 3rd residue is generated by steric requirements of the close packing of the three supercoiled polypeptide chains near a central axis. The collagen amino acid sequence is also characterized by a high frequency of proline and hydroxyproline (Hyp 4 ) in the X and Y positions, respectively, which serves to stabilize the polyproline II-like helix of the individual chains. The collagen triple helix is the defining motif of all extracellular matrix proteins classified as collagens and is also found as a domain in various other proteins, including C1q, macrophage scavenger receptor, collagenous tail of asymmetric acetylcholinesterase, and the bacterial proteins Streptococcus pyogenes scl and Bacillus anthraces bcl1 (5-9).Although the (Gly-X-Y) n repeating sequence is a requirement to form a collagen triple helix, this pattern is perfectly maintained in some collagen domains but not in others (9). The most abundant collagens are those found in fibrils with a 670 Å axial period (types I, II, III, V, XI), and these all maintain a precise Gly-X-Y repeat throughout their ϳ1000-residue triple helix domain. The replacement of even one Gly by anot...

Missense mutations in the collagen triple-helix that replace one of the required Gly residues in the (Gly-Xaa-Yaa)(n)() repeating sequence have been implicated in various disorders. Although most hereditary collagen disorders are rare, a common occurrence of a Gly replacement mutation is found in the collagenous domain of mannose binding lectin (MBL). A Gly --> Asp mutation at position 54 in MBL is found at a frequency as high as 30% in certain populations and leads to increased susceptibility to infections. The structural and energetic consequences of this mutation are investigated by comparing a triple-helical peptide containing the N-terminal Gly-X-Y units of MBL with the homologous peptide containing the Gly to Asp replacement. The mutation leads to a loss of triple-helix content but only a small decrease in the stability of the triple-helix (DeltaT(m) approximately 2 degrees C) and no change in the calorimetric enthalpy. NMR studies on specifically labeled residues indicate the portion of the peptide C-terminal to residue 54 is in a highly ordered triple-helix in both peptides, while residues N-terminal to the mutation site have a weak triple-helical signal in the parent peptide and are completely disordered in the mutant peptide. These results suggest that the N-terminal triplet residues are contributing little to the stability of this peptide, a hypothesis confirmed by the stability and enthalpy of shorter peptides containing only the region C-terminal to the mutation site. The Gly to Asp replacement at position 54 in MBL occurs at the boundary of a highly stable triple-helix region and a very unstable sequence. The junctional position of this mutation minimizes its destabilizing effect, in contrast with the significant destabilization seen for Gly replacements in peptides modeling collagen diseases.