Anton V. Persikov

Molecular Structure of the Collagen Triple Helix

The molecular conformation of the collagen triple helix confers strict amino acid sequence constraints, requiring a (Gly-X-Y)(n) repeating pattern and a high content of imino acids. The increasing family of collagens and proteins with collagenous domains shows the collagen triple helix to be a basic motif adaptable to a range of proteins and functions. Its rodlike domain has the potential for various modes of self-association and the capacity to bind receptors, other proteins, GAGs, and nucleic acids. High-resolution crystal structures obtained for collagen model peptides confirm the supercoiled triple helix conformation, and provide new information on hydrogen bonding patterns, hydration, sidechain interactions, and ligand binding. For several peptides, the helix twist was found to be sequence dependent, and such variation in helix twist may serve as recognition features or to orient the triple helix for binding. Mutations in the collagen triple-helix domain lead to a variety of human disorders. The most common mutations are single-base substitutions that lead to the replacement of one Gly residue, breaking the Gly-X-Y repeating pattern. A single Gly substitution destabilizes the triple helix through a local disruption in hydrogen bonding and produces a discontinuity in the register of the helix. Molecular information about the collagen triple helix and the effect of mutations will lead to a better understanding of function and pathology.

Prediction of Collagen Stability from Amino Acid Sequence

An algorithm was derived to relate the amino acid sequence of a collagen triple helix to its thermal stability. This calculation is based on the triple helical stabilization propensities of individual residues and their intermolecular and intramolecular interactions, as quantitated by melting temperature values of host-guest peptides. Experimental melting temperature values of a number of triple helical peptides of varying length and sequence were successfully predicted by this algorithm. However, predicted Tm values are significantly higher than experimental values when there are strings of oppositely charged residues or concentrations of like charges near the terminus. Application of the algorithm to collagen sequences highlights regions of unusually high or low stability, and these regions often correlate with biologically significant features. The prediction of stability from sequence indicates an understanding of the major forces maintaining this protein motif. The use of highly favorable KGE and KGD sequences is seen to complement the stabilizing effects of imino acids in modulating stability and may become dominant in the collagenous domains of bacterial proteins that lack hydroxyproline. The effect of single amino acid mutations in the X and Y positions can be evaluated with this algorithm. An interactive collagen stability calculator based on this algorithm is available online.

Self-association of Collagen Triple Helix Peptides into Higher Order Structures *□

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 β-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 nucleation-growth 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, (Pro-Hyp-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.

Collagen model peptides: Sequence dependence of triple‐helix stability

The triple helix is a specialized protein motif, found in all collagens as well as in noncollagenous proteins involved in host defense. Peptides will adopt a triple-helical conformation if the sequence contains its characteristic features of Gly as every third residue and a high content of Pro and Hyp residues. Such model peptides have proved amenable to structural studies by x-ray crystallography and NMR spectroscopy, suitable for thermodynamic and kinetic analysis, and a valuable tool in characterizing the binding activities of the collagen triple helix. A systematic approach to understanding the amino acid sequence dependence of the collagen triple helix has been initiated, based on a set of host-guest peptides of the form, (Gly-Pro-Hyp)(3)-Gly-X-Y-(Gly-Pro-Hyp)(4). Comparison of their thermal stabilities has led to a propensity scale for the X and Y positions, and the additivity of contributions of individual residues is now under investigation. The local and global stability of the collagen triple helix is normally modulated by the residues in the X and Y positions, with every third position occupied by Gly in fibril-forming collagens. However, in collagen diseases, such as osteogenesis imperfecta, a single Gly may be substituted by another residue. Host-guest studies where the Gly is replaced by various amino acids suggest that the identity of the residue in the Gly position affects the degree of destabilization and the clinical severity of the disease.

Unstable molecules form stable tissues

Collagens are major structural proteins in the extracellular matrix, making up about one-third of protein mass in higher animals. In addition to their sheer bulk, this protein family is of interest because of their diversity of structural and morphogenetic roles and the attribution of an increasing number of hereditary diseases to mutations in collagens (1–4). All collagens have a distinctive molecular conformation: a triple-helix composed of three supercoiled polyproline II-like helical chains (5–7). This triple-helical conformation places strict constraints on amino acid sequence, requiring Gly as every third residue and a high content of proline and hydroxyproline residues. There are more than 20 distinct genetic types of collagens, and the most abundant are types I, II, and III, found in fibrils with a characteristic 67-nm axial period (1). Type I collagen, a heterotrimer composed of two α1(I) chains and one α2(I) chain, forms the prominent fibrils in tendon, bone, and cornea, whereas type III collagen, a disulfide-linked homotrimer, is found together with type I in fibrils of blood vessels and skin. These fibril-forming collagens are synthesized in a procollagen form, with globular propeptides on each end of a central triple-helix (Fig. 1; ref. 3). Self-association and disulfide cross-linking of three C-propeptides are responsible for the initial events of chain selection and trimer formation, whereas subsequent events include nucleation and zipper-like folding of the triple-helix domain (8). After cleavage of the propeptides, the rod-like triple-helical molecules in the matrix self-associate in a staggered array, forming fibrils and interacting with other matrix molecules to provide the strength, flexibility, or compression required for each tissue. Collagen fibers are inherently stable structures, having lifetimes of at least 6 months, and often much longer. Turnover is accomplished through a specialized family of tightly regulated matrix metalloproteinases, because triple-helices are resistant …

Triple-helix propensity of hydroxyproline and fluoroproline: comparison of host-guest and repeating tripeptide collagen models.

Peptide models have proved important in defining the structural features of the collagen triple-helix. Some models are based on multiple repeats of a given tripeptide unit, while a host-guest design includes an individual tripeptide unit substituted within a constant repeating Pro-Hyp-Gly framework. In the present study, proline, hydroxyproline, and fluoroproline residues are incorporated in X- or Y-positions of a guest triplet in the host-guest peptide design. All host-guest peptides, including Hyp-Pro-Gly, formed stable triple-helices, even though a triple-helix cannot be formed by (Hyp-Pro-Gly)10. The order of stability Pro-Hyp-Gly > Pro-Pro-Gly > Hyp-Pro-Gly remains the same in all models, while the Pro-Flp-Gly is very stabilizing in a repeating context but destabilizing in a host-guest context. The range of thermal stabilities and calorimetric enthalpies is very small among the five host-guest peptides, consistent with the concept that the effect of one Xaa-Yaa-Gly tripeptide unit in the host-guest system would be less than the much larger variations when there are 10 repeating units. However, a simple additive model based on host-guest peptides predicts a greater stability than experimentally observed. The difference in stability contributions of the same tripeptide unit in host-guest versus repeating tripeptide systems illustrates the impact of sequence environment on stability, and factors that play a role include ring puckering as a consequence of electron inductive effects, residual monomer structure, and native state hydration networks.

Designed coiled coils promote folding of a recombinant bacterial collagen.

Collagen triple helices fold slowly and inefficiently, often requiring adjacent globular domains to assist this process. In the Streptococcus pyogenes collagen-like protein Scl2, a V domain predicted to be largely α-helical, occurs N-terminal to the collagen triple helix (CL). Here, we replace this natural trimerization domain with a de novo designed, hyperstable, parallel, three-stranded, α-helical coiled coil (CC), either at the N terminus (CC-CL) or the C terminus (CL-CC) of the collagen domain. CD spectra of the constructs are consistent with additivity of independently and fully folded CC and CL domains, and the proteins retain their distinctive thermal stabilities, CL at ∼37 °C and CC at >90 °C. Heating the hybrid proteins to 50 °C unfolds CL, leaving CC intact, and upon cooling, the rate of CL refolding is somewhat faster for CL-CC than for CC-CL. A construct with coiled coils on both ends, CC-CL-CC, retains the ∼37 °C thermal stability for CL but shows less triple helix at low temperature and less denaturation at 50 °C. Most strikingly however, in CC-CL-CC, the CL refolds slower than in either CC-CL or CL-CC by almost two orders of magnitude. We propose that a single CC promotes folding of the CL domain via nucleation and in-register growth from one end, whereas initiation and growth from both ends in CC-CL-CC results in mismatched registers that frustrate folding. Bioinformatics analysis of natural collagens lends support to this because, where present, there is generally only one coiled-coil domain close to the triple helix, and it is nearly always N-terminal to the collagen repeat.

Fluorescence Determination of Tryptophan Side-Chain Accessibility and Dynamics in Triple-Helical Collagen-Like Peptides

We report tryptophan fluorescence measurements of emission intensity, iodide quenching, and anisotropy that describe the environment and dynamics at X and Y sites in stable collagen-like peptides of sequence (Gly-X-Y)(n). About 90% of tryptophans at both sites have similar solvent exposed fluorescence properties and a lifetime of 8.5-9 ns. Analysis of anisotropy decays using an associative model indicates that these long lifetime populations undergo rapid depolarizing motion with a 0.5 ns correlation time; however, the extent of fast motion at the Y site is considerably less than the essentially unrestricted motion at the X site. About 10% of tryptophans at both sites have a shorter ( approximately 3 ns) lifetime indicating proximity to a protein quenching group; these minor populations are immobile on the peptide surface, depolarizing only by overall trimer rotation. Iodide quenching indicates that tryptophans at the X site are more accessible to solvent. Side chains at X sites are more solvent accessible and considerably more mobile than residues at Y sites and can more readily fluctuate among alternate intermolecular interactions in collagen fibrils. This fluorescence analysis of collagen-like peptides lays a foundation for studies on the structure, dynamics, and function of collagen and of triple-helical junctions in gelatin gels.

The Generalized Heat Capacity/Amylose Content Function for Barley and Maize Starches

The heat capacity values of 1 % aqueous dispersions of barley and maize starches with different amylose content (1—35 %), were determined. It was shown that a rise in amylose content leads to an increase of heat capacity values. The heat capacity values were calculated per 1 % change of amylose content in barley and maize starches. The generalized function describing changes of heat capacity values versus amylose content for barley and maize starches has been established.

Heat capacity values of 1% aqueous dispersions for native maize starches with different amylose content

The heat capacities (HC) of 1% aqueous dispersions of native maize starches with different amylose content were determined by using high sensitivity differential scanning microcalorimetry (HSDSC). The application of an additive equation, taking into account the contributions of different ordered structures (B-, B * - and V-types) to the heat capacity of the high amylose maize starches, allows to describe the changes in the heat capacity of the maize starches at an increase in amylose content from 0% to 70%. The heat capacity values per 1% change both of B- and V-type structures in high amylose starches have been determined. Additionally, the calculations showed that at an amylose content in maize starches equal to 0%, the heat capacity values for A- and B * -type structures are, 164 Jmol -1 K -1 and 186 Jmol -1 K -1 , respectively. The relationship between heat capacity values and structural features of maize starches is discussed.