Presence of a thermostable domain in the helical part of the type I collagen molecule and its role in the mechanism of triple helix folding.

A study has been done of the effect of neutral salts (NaCl and CaCl2) on the mechanism of type I collagen triple helix folding and unfolding in concentrated acetic acid solutions (2-8.8 M). It is shown that in these conditions, thermoabsorption and secondary structure change in heated solutions proceed in two consecutive stages. Salts exert a different destabilizing effect on different sites of the macromolecule, promoting the detection of a thermostable domain. The presence of a thermostable domain permits one to carry out reversible denaturation of collagen and to study the mechanism of the triple helix folding. Proceeding from the mechanism of the triple helix folding, an assumption has been made on the localization of the thermostable domain and its biological role.     

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.     

Synthesis and triplex binding properties of oligonucleotides containing a novel nucleobase

The thiazolo-indole compound 1 bearing the complementary donor-acceptor-donor sites (dad) was designed for specific recognition of an AT inverted base pair in pyrimidine triple helix motif. It was successfully incorporated into 14-mer oligonucleotide using a serinol unit as sugar derivative. The triple helix hybridization studies were examined by means of thermal denaturation experiments with a 26-mer DNA duplex containing the AT inverted base pair.     

Binding of netropsin to a DNA triple helix.

The interaction of netropsin, a minor groove binding drug, with T-A-T triple helix and A-T double helix was studied using circular dichroism spectroscopy and thermal denaturation. The triple helix was made by an oligonucleotide (dA)12-x-(dT)12-x-(dT)12, where x is a hexaethylene glycol chain bridged between the 3' phosphate of one strand and the 5' phosphate of the following strand. This oligonucleotide is able to fold back on itself to form a very stable triplex. Changing the conditions allows the same oligonucleotide in a duplex form with a (dT)12 dangling arm. Circular dichroism spectroscopy demonstrates that netropsin can bind to the triple helical structure. Spectral analysis shows that the bound drug exhibits a conformation and an environment similar in double-stranded and in triple-stranded structure. However, the binding constant to the triple-stranded structure is found smaller than the binding constant to the double-stranded one. Thermal denaturation experiments demonstrate that netropsin destabilizes the triplex whereas it stabilizes the duplex.     

Synthesis and Triplex Binding Properties of Oligonucleotides Containing a Novel Nucleobase

Abstract

The thiazolo-indole compound 1 bearing the complementary donor-acceptor-donor sites (dad) was designed for specific recognition of an AT inverted base pair in pyrimidine triple helix motif. It was successfully incorporated into 14-mer oligonucleotide using a serinol unit as sugar derivative. The triple helix hybridization studies were examined by means of thermal denaturation experiments with a 26-mer DNA duplex containing the AT inverted base pair.     

The C-propeptide domain of procollagen can be replaced with a transmembrane domain without affecting trimer formation or collagen triple helix folding during biosynthesis.

The folding and assembly of procollagen occurs within the cell through a series of discrete steps leading to the formation of a stable trimer consisting of three distinct domains: the N-propeptide, the C-propeptide and the collagen triple helix flanked at either end by short telopeptides. We have established a semi-permeabilized cell system which allows us to study the initial stages in the folding and assembly of procollagen as they would occur in the intact cell. By studying the folding and assembly of the C-propeptide domain in isolation, and a procollagen molecule which lacks the C-propeptide, we have shown that this domain directs the initial association event and is required to allow triple helix formation. However, the essential function of this domain does not include triple helix nucleation or alignment, since this can occur when the C-propeptide is substituted with a single transmembrane domain. Also the telopeptide region is not involved in triple helix nucleation; however, a minimum of two hydroxyproline-containing Gly-X-Y triplets at the C-terminal end of the triple helix are required for nucleation to occur. Thus, the C-propeptide is required solely to ensure association of the monomeric chains; once these are brought together, the triple helix is able to nucleate and fold to form a correctly aligned triple helix.     

Hydroxyproline content determines the denaturation temperature of chick tendon collagen

A series of nine procollagen samples in which the hydroxyproline content varied from <1% to 44% of the total imino acids was prepared by incubating embryonic chick tendon cells with varying concentrations of α,α′-dipyridyl, an inhibitor of proline hydroxylase. The thermal stability of these procollagen preparations was then investigated by using pepsin digestion at different temperatures as an enzymatic probe of conformation. Using this technique, the denaturation temperature of the procollagen was found to be directly proportional to the hydroxyproline content. A denaturation temperature of 23.5 °C was found for the unhydroxylated procollagen and 37.9 °C for fully hydroxylated procollagen. These results suggest that hydroxyproline is crucial to the thermal stability of the collagen triple helix. They also imply that unhydroxylated molecules are not triple helical within the cell at 37 °C and that triple helix formation may be necessary for normal secretion.     

Influence of tertiary structure domain properties on the functionality of apolipoprotein A-I

The tertiary structure of apolipoprotein (apo) A-I and the contributions of structural domains to the properties of the protein molecule are not well defined. We used a series of engineered human and mouse apoA-I molecules in a range of physical-biochemical measurements to address this issue. Circular dichroism measurements of alpha-helix thermal unfolding and fluorescence spectroscopy measurements of 8-anilino-1-napthalenesulfonic acid binding indicate that removal of the C-terminal 54 amino acid residues from human and mouse apoA-I has similar effects; the molecules are only slightly destabilized, and there is a decrease in hydrophobic surface exposure. These results are consistent with both human and mouse apoA-I adopting a two-domain tertiary structure, comprising an N-terminal antiparallel helix bundle domain and a separate less ordered C-terminal domain. Mouse apoA-I is significantly less resistant than human apoA-I to thermal and chemical denaturation; the midpoint of thermal unfolding of mouse apoA-I at 45 degrees C is 15 degrees C lower and the midpoint of guanidine hydrochloride denaturation (D1/2) occurs at 0.5 M as compared to 1.0 M for human apoA-I. These differences reflect the overall greater stability of the helix bundle formed by residues 1-189 in human apoA-I. Measurements of the heats of binding to egg phosphatidylcholine (PC) small unilamellar vesicles and the kinetics of solubilization of dimyristoyl PC multilamellar vesicles indicate that the more stable human helix bundle interacts poorly with lipids as compared to the equivalent mouse N-terminal domain. The C-terminal domain of human apoA-I is much more hydrophobic than that of mouse apoA-I; in the lipid-free state the human C-terminal domain (residues 190-243) is partially alpha-helical and undergoes cooperative unfolding (D1/2 = 0.3 M) whereas the isolated mouse C-terminal domain (residues 187-240) is disordered in dilute solution. The human C-terminal domain binds to lipid surfaces much more avidly than the equivalent mouse domain. Human and mouse apoA-I have very different tertiary structure domain contributions for achieving functionality. It is clear that the stability of the N-terminal helix bundle, and the hydrophobicity and alpha-helix content of the C-terminal domain, are critical factors in determining the overall properties of the apoA-I molecule.     

The Cysteine-rich Region of Type VII Collagen Is a Cystine Knot with a New Topology

Collagens are a group of extracellular matrix proteins with essential functions for skin integrity. Anchoring fibrils are made of type VII collagen (Col7) and link different skin layers together: the basal lamina and the underlying connective tissue. Col7 has a central collagenous domain and two noncollagenous domains located at the N and C terminus (NC1 and NC2), respectively. A cysteine-rich region of hitherto unknown function is located at the transition of the NC1 domain to the collagenous domain. A synthetic model peptide of this region was investigated by CD and NMR spectroscopy. The peptide folds into a collagen triple helix, and the cysteine residues form disulfide bridges between the different strands. The eight cystine knot topologies that are characterized by exclusively intermolecular disulfide bridges have been analyzed by molecular modeling. Two cystine knots are energetically preferred; however, all eight disulfide bridge arrangements are essentially possible. This novel cystine knot is present in type IX collagen, too. The conserved motif of the cystine knot is CX₃CP. The cystine knot is N-terminal to the collagen triple helix in both collagens and therefore probably impedes unfolding of the collagen triple helix from the N terminus.     

Molecular Modelling Study of the Netropsin Complexation With a Nucleic Acid Triple Helix

Abstract

A detailed molecular mechanical study has been made on the complexes of netropsin with the double stranded oligonucleotide (dA)₁₂.(dT)₁₂ and with the triple helix (dA)₁₂.(dT)₁₂.(dT)₁₂. The complexes were built using computer graphics and energy refined using JUMNA program. In agreement with circular dichroïsm experiments we have shown that 3 netropsins can bind the minor grooves of the triple helix and of the double helix. The groove geometry in the duplex and in the triplex is very similar. However a detailed analysis of the energetic terms shows, in agreement with thermal denaturation studies, that the affinity of netropsin toward the double helices is larger than towards triple helices.     

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.     

Berenil Complexation with a Nucleic Acid Triple Helix

Abstract

The interaction of berenii molecule, a minor groove binding drug, with T-A-T triple helix and A-T double helix was studied using circular dichroism spectroscopy and thermal denaturation. The triple helix was made by an oligonucleotide (dA)₁₂?x-(dT)₁₂?x-(dT)₁₂, where x is a hexaethylene glycol chain bridged between the 3′ phosphate of one strand and the 5′ phosphate of the following strand. This oligonucleotide is able to fold back on itself to form a very stable triplex. Circular dichroism spectroscopy demonstrates that berenil can bind to the triple helical structure. Spectral analysis shows that in the same ionic strength the drug bound to a double-stranded structure exhibits a conformation and an environment close to those observed in triple-stranded structure. The influence of the ionic strength on the interaction between the berenil molecule and the 36-mer is clearly demonstrated. We showed that when no NaCl salt is added in the buffer the triplex form of (dA)₁₂?x-(dT)₁2-x-(dT)₁₂ is stabilized by berenil whereas it is destabilized slightly by the dye when NaCl concentration is 1 M.     

Microcalorimetric and electron spin resonance study of the influence of UV radiation on collagen

It has been shown by microcalorimetry that UV-irradiation cardinally alters the temperature dependence of heat capacity of a collagen solution and decreases the enthalpy of collagen heat denaturation. By using the method of electron spin resonance (ESR), it was found that the primary products of UV-irradiated acid-soluble collagen are the atomic hydrogen and the anion radical of acetic acid. The latter, under the influence of long-wavelength UV light, is transformed into the methyl radical, which interacts with acetic acid to produce acetic acid radical. The above free radicals interact with the collagen molecule, as a result of which seven superfine components with the split of deltaH = 1.13 mT are obtained in the ESR spectrum. It is assumed that this spectrum is related to the free radical that occurred in the proline residue of the collagen molecule. In this particular case, this is a major structural defect in the triple helix of collagen, which results in instability of the macromolecule.     

Noncollagenous region of the streptococcal collagen‐like protein is a trimerization domain that supports refolding of adjacent homologous and heterologous collagenous domains

Proper folding of the (Gly‐Xaa‐Yaa)_n sequence of animal collagens requires adjacent N‐ or C‐terminal noncollagenous trimerization domains which often contain coiled‐coil or beta sheet structure. Collagen‐like proteins have been found recently in a number of bacteria, but little is known about their folding mechanism. The Scl2 collagen‐like protein from Streptococcus pyogenes has an N‐terminal globular domain, designated V_sp, adjacent to its triple‐helix domain. The V_sp domain is required for proper refolding of the Scl2 protein in vitro. Here, recombinant V_sp domain alone is shown to form trimers with a significant α‐helix content and to have a thermal stability of T_m = 45°C. Examination of a new construct shows that the V_sp domain facilitates efficient in vitro refolding only when it is located N‐terminal to the triple‐helix domain but not when C‐terminal to the triple‐helix domain. Fusion of the V_sp domain N‐terminal to a heterologous (Gly‐Xaa‐Yaa)_n sequence from Clostridium perfringens led to correct folding and refolding of this triple‐helix, which was unable to fold into a triple‐helical, soluble protein on its own. These results suggest that placement of a functional trimerization module adjacent to a heterologous Gly‐Xaa‐Yaa repeating sequence can lead to proper folding in some cases but also shows specificity in the relative location of the trimerization and triple‐helix domains. This information about their modular nature can be used in the production of novel types of bacterial collagen for biomaterial applications.     

Sequence-specific recognition of collagen triple helices by the collagen-specific molecular chaperone HSP47

HSP47 is a molecular chaperone that plays an unknown role during the assembly and transport of procollagen. Our previous studies showed that, unlike most chaperones, HSP47 interacts with a correctly folded substrate. We suggested that HSP47 either stabilizes the correctly folded collagen helix from heat denaturation or prevents lateral aggregation prior to its transport from the endoplasmic reticulum. In this study we have addressed the role of triple helix stability in the binding of HSP47 to procollagen by expressing procollagen molecules with differing thermal stabilities and analyzing their ability to interact with HSP47 within the endoplasmic reticulum. Our results show that HSP47 interacts with thermostable procollagen molecules, suggesting that helix stabilization is not the primary function of HSP47 and that the interaction of HSP47 with procollagen depends upon the presence of a minimum of one Gly-X-Arg triplet within the triple helical domain. Interestingly, procollagen chains containing high proportions of stabilizing triplets formed triple helices and interacted with HSP47 even in the absence of proline hydroxylation, demonstrating that recognition does not depend upon this modification. Our results support the view that HSP47 functions early in the secretory pathway by preventing the lateral aggregation of procollagen chains.     

A microcalorimetric and electron spin resonance study of the influence of UV radiation on collagen

It was demonstrated microcalorimetrically that UV radiation cardinally changed the behavioral pattern of the temperature dependence of heat capacity of a collagen solution and decreased the enthalpy of collagen denaturation. It was discovered by electron spin resonance (ESR) that the primary products of UV-irradiated acid-soluble collagen were atomic hydrogen and the anion radical of acetic acid. The latter, when exposed to long-wavelength UV light, converted into the methyl radical, which interacted with acetic acid to produce the acetic acid radical. These free radicals interacted with the collagen molecule, leading to the appearance of seven superfine components with the split ΔH = 1.13 mT in the ESR spectrum. It was assumed that this spectrum is related to the free radical that occurred in the proline residue of the collagen molecule, which, in this particular case, was the major defect in the triple helix of collagen that caused instability of the macromolecule.     

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.     

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.     

Molecular mechanism of alpha 1(I)-osteogenesis imperfecta/Ehlers-Danlos syndrome: unfolding of an N-anchor domain at the N-terminal end of the type I collagen triple helix

We demonstrate that 85 N-terminal amino acids of the alpha1(I) chain participate in a highly stable folding domain, acting as the stabilizing anchor for the amino end of the type I collagen triple helix. This anchor region is bordered by a microunfolding region, 15 amino acids in each chain, which include no proline or hydroxyproline residues and contain a chymotrypsin cleavage site. Glycine substitutions and amino acid deletions within the N-anchor domain induce its reversible unfolding above 34 degrees C. The overall triple helix denaturation temperature is reduced by 5-6 degrees C, similar to complete N-anchor removal. N-propeptide partially restores the stability of mutant procollagen but not sufficiently to prevent N-anchor unfolding and a conformational change at the N-propeptide cleavage site. The ensuing failure of N-proteinase to cleave at the misfolded site leads to incorporation of pN-collagen into fibrils. Similar, but weaker, effects are caused by G88E substitution in the adjacent triplet, which appears to alter N-anchor structure as well. As in Ehlers-Danlos syndrome (EDS) VIIA/B, fibrils containing pN-collagen are thinner and weaker causing EDS-like laxity of large and small joints and paraspinal ligaments. However, distinct structural consequences of N-anchor destabilization result in a distinct alpha1(I)-osteogenesis imperfecta (OI)/EDS phenotype.     

Study of the structural properties of recombinant gamma-interferon by circular dichroism and differential scanning microcalorimetry]

Recombinant human gamma-interferon is dimeric in solution at pH 7-4 as revealed by analytical gel-filtration. It was shown by circular dichroism that decreasing pH to 5.0 does not affect the secondary and tertiary structures of gamma-interferon macromolecule. It was established that heat denaturation process of gamma-interferon obeys the two-state transition model and can be described as the first-order reversible reaction. Temperature dependence of the denaturation-renaturation rate constants was shown to be consistent with the Arrhenius law. The equilibrium value of the denaturation temperature was found. Effective enthalpy of denaturation was determined both by thermodynamic and kinetic approaches. The data obtained showed that in the pH range 7-4 the dimeric IFN-gamma structure may be considered as a single cooperative thermodynamic domain. Thus, it may be concluded that gamma-interferon dimerization is necessary for the existence of the corresponding tertiary structure of the macromolecule.