The role of cystine knots in collagen folding and stability,part II. Conformational properties of (Pro-Hyp-Gly)n model trimers with N- and C-terminal collagen type III cystine knots

In mature collagen type III the homotrimer is C-terminally cross-linked by an interchain cystine knot consisting of three disulfide bridges of unknown connectivity. This cystine knot with two adjacent cysteine residues on each of the three alpha chains has recently been used for the synthesis and expression of model homotrimers. To investigate the origin of correct interchain cysteine pairings, (Pro-Hyp-Gly)(n) peptides of increasing triplet number and containing the biscysteinyl sequence C- and N-terminally were synthesised. The possibilities were that this origin may be thermodynamically coupled to the formation of the collagen triple helix as happens in the oxidative folding of proteins, or it could represent a post-folding event. Only with five triplets, which is known to represent the minimum number for self-association of collagenous peptides into a triple helix, air-oxidation produces the homotrimer in good yields (70 %), the rest being intrachain oxidised monomers. Increasing the number of triplets has no effect on yield suggesting the formation of kinetically trapped intermediates, which are not reshuffled by the glutathione redox buffer. N-terminal incorporation of the cystine knot is significantly less efficient in the homotrimerisation step and also in terms of triple-helix stabilisation. Compared to an artificial C-terminal cystine knot consisting of two interchain disulfide bridges, the collagen type III cystine knot produces collagenous homotrimers of remarkably high thermostability, although the concentration-independent refolding rates are not affected by the type of disulfide bridging. Since the natural cystine knot allows ready access to homotrimeric collagenous peptides of significantly enhanced triple-helix thermostability it may well represent a promising approach for the preparation of collagen-like innovative biomaterials. Conversely, the more laborious regioselectively formed artificial cystine knot still represents the only synthetic strategy for heterotrimeric collagenous peptides.     

Photocontrol of the collagen triple helix: synthesis and conformational characterization of bis-cysteinyl collagenous peptides with an azobenzene clamp

For the photomodulation of the collagen triple helix with an azobenzene clamp, we investigated various collagenous peptides consisting of ideal (Gly-Pro-Hyp) repeats and containing cysteine residues in various positions for a side chain-to-side chain crosslink with a suitable chromophore derivative. Comparative conformational analysis of these cysteine peptides indicated an undecarepeat peptide with two cysteine residues located in the central portion in i and i+7 positions and flanked by (Gly-Pro-Hyp) repeat sequences as the most promising for the cross-bridging experiments. In aqueous alcoholic solution the azobenzene-undecarepeat peptide formed a stable triple helix in equilibrium with the monomeric species as a trans-azobenzene isomer, whereas photoisomerization to the cis isomer leads to unfolding of at least part of the triple helix. Furthermore, the residual supercoiled structure acts like an intermolecular knot, thus making refolding upon cis-to-trans isomerization a concentration-independent fast event. Consequently, these photoswitchable collagenous systems should be well suited for time-resolved studies of folding/unfolding of the collagen triple helix under variable thermodynamic equilibria.     

Cyclotriveratrylene (CTV) as a new chiral triacid scaffold capable of inducing triple helix formation of collagen peptides containing either a native sequence or Pro-Hyp-Gly repeats

A new triacid scaffold is described based on the cone-shaped cyclotriveratrylene (CTV) molecule that facilitates the triple helical folding of peptides containing either a unique blood platelet binding collagen sequence or collagen peptides composed of Pro-Hyp-Gly repeats. The latter were synthesized by segment condensation using Fmoc-Pro-Hyp-Gly-OH. Peptides were coupled to this CTV scaffold and also coupled to the Kemp's triacid (KTA) scaffold. After assembly of peptide H-Gly-[Pro-Hyp-Gly]2-Phe-Hyp-Gly-Glu(OAll)-Arg-Gly-Val-Glu (OAll)-Gly-[Pro-Hyp-Gly]2-NH2 (13) by an orthogonal synthesis strategy to both triacid scaffolds, followed by deprotection of the allyl groups, the molecular constructs spontaneously folded into a triple helical structure. In contrast, the non-assembled peptides did not. The melting temperature (Tm) of (+/-) CTV[CH2C(O)N(H)Gly-[Pro-Hyp-Gly]2-Phe-Hyp-Gly-Glu-Arg-Gly-Val-Glu-Gly- [Pro-Hyp-Gly]2-NH2]3 (14) is 19 degrees C, whereas KTA[Gly-Gly-[Pro-Hyp-Gly]2-Phe-Hyp-Gly-Glu-Arg-Gly-Val-Glu-Gly- [Pro-Hyp-Gly]2-NH2]3 (15) has a Tm of 20 degrees C. Thus, it was shown for the first time that scaffolds were also effective in stabilizing the triple helix of native collagen sequences. The different stabilizing properties of the two CTV enantiomers could be measured after coupling of racemic CTV triacid to the collagen peptide, and subsequent chromatographic separation of the diastereomers. After assembly of the two chiral CTV scaffolds to the model peptide H-Gly-Gly-(Pro-Hyp-Gly)5-NH2 (24), the (+)-enantiomer of CTV 28b was found to serve as a better triple helix-inducing scaffold than the (-)-enantiomer 28a. In addition to an effect of the chirality of the CTV scaffold, a certain degree of flexibility between the CTV cone and the folded peptide was also shown to be of importance. Restricting the flexibility from two to one glycine residues resulted in a significant difference between the two collagen mimics 20a and 20b, whereas the difference was only slight when two glycine residues were present between the CTV scaffold and the peptide sequence in collagen mimics 30a and 30b.     

Complete Mapping of a Cystine Knot and Nested Disulfides of Recombinant Human Arylsulfatase A by Multi-Enzyme Digestion and LC-MS Analysis Using CID and ETD

Cystine knots or nested disulfides are structurally difficult to characterize, despite current technological advances in peptide mapping with high-resolution liquid chromatography coupled with mass spectrometry (LC-MS). In the case of recombinant human arylsulfatase A (rhASA), there is one cystine knot at the C-terminal, a pair of nested disulfides at the middle, and two out of three unpaired cysteines in the N-terminal region. The statuses of these cysteines are critical structure attributes for rhASA function and stability that requires precise examination. We used a unique approach to determine the status and linkage of each cysteine in rhASA, which was comprised of multi-enzyme digestion strategies (from Lys-C, trypsin, Asp-N, pepsin, and PNGase F) and multi-fragmentation methods in mass spectrometry using electron transfer dissociation (ETD), collision induced dissociation (CID), and CID with MS³ (after ETD). In addition to generating desired lengths of enzymatic peptides for effective fragmentation, the digestion pH was optimized to minimize the disulfide scrambling. The disulfide linkages, including the cystine knot and a pair of nested cysteines, unpaired cysteines, and the post-translational modification of a cysteine to formylglycine, were all determined. In the assignment, the disulfide linkages were Cys138–Cys154, Cys143–Cys150, Cys282–Cys396, Cys470–Cys482, Cys471–Cys484, and Cys475–Cys481. For the unpaired cysteines, Cys20 and Cys276 were free cysteines, and Cys51 was largely converted to formylglycine (>70 %). A successful methodology has been developed, which can be routinely used to determine these difficult-to-resolve disulfide linkages, ensuring drug function and stability.      

Controlling the morphology of metal-promoted higher ordered assemblies of collagen peptides with varied core lengths

Self-assembling peptides have become an important subclass of next-generation biomaterials. In particular, materials that mimic the properties of collagen have received considerable attention due to the unique properties of natural collagen. Previous peptide-based designs have been successful in generating structures with morphological properties that were primarily determined by the type of self-assembling mechanism. Herein we demonstrate the metal ion-promoted, supramolecular assembly of collagen-based peptide triple helices into distinct morphologies that are controlled by defining the number of Pro-Hyp-Gly repeating units. We synthesized and characterized collagen-based peptides that incorporated either 5, 7, 9, or 11 Pro-Hyp-Gly repeating units. We found that the number of repeating units, and the resulting stability of the collagen triple helix, is intimately linked with the types of assemblies formed. For instance, collagen peptides that did not form a stable triple helix, such as NCoH5, did not participate in supramolecular assembly with added metal ions. Collagen peptides that formed stable triple helices, such as NCoH11, resulted in microsaddle structures with metal-promoted assembly, whereas a highly cross-linked, three-dimensional mesh formed with NCoH7, albeit at a higher metal ion concentration. These data provide evidence that triple helix formation is required for efficient metal-triggered assembly to the observed microstructures.     

The attempted synthesis of open-chain asymmetric cystine peptides by thio-induced fragmentation of sulfonyl thiocarbonates. Insulin fragments with intact disulfide bridges A20-B19

The thiol-induced fragmentations of sulfenyl thiocarbonates (R? S? S? CO? OCH₃) leading to mixed disulfides was applied to the synthesis of open-chain asymmetrical cystine peptides. Various fragments of insulin containing the disulfide bridge between A²⁰ and B¹⁹ were prepared by this method.     

Measurement of urinary cystine and cysteinyl-penicillamine in patients with cystinuria

A high-performance liquid chromatographic method with electrochemical detection (LC/EC) was developed to measure cystine and cysteinyl-penicillamine disulfide in the urine of patients screened or treated for cystinuria. Urine was acidified, centrifuged to remove urinary protein, diluted and injected. The disulfides were separated on a reversed-phase column, reduced at the upstream electrode of a dual electrochemical detector with gold-mercury amalgam (Au/Hg) electrodes and the resultant thiols measured at the downstream electrode. The sample preparation is simple, the analysis rapid, specimens can be easily batched and the specificity of the method is better than those of two other separative procedures with which it was compared. The coefficient of variation for cystine in cystinuric urine is 6.7%, 5.5% and 3.2% for levels of 0.09, 0.52 and 1.02 mmol/l respectively, and for cysteinyl-penicillamine disulfide 2.6% and 7.5% for levels of 0.45 and 0.98 mmol/l respectively. Urine for analysis of these disulfides should not be collected within 24 hours of administration of the radiopaque agent diatrizoate but no other interference to the assay has been noted. This method is suitable as a screen for cystinuria in patients with renal tract calculi, for ongoing monitoring of cystinuric patients and to check patient compliance with d-penicillamine therapy.     

Resonant dissociative electron attachments to cysteine and cystine

Shape-resonant electron attachments to cysteine and cystine and the subsequent dissociation dynamics are investigated with the single-center expansion potential scattering calculations. Selectivity of the direct bond cleavage at a given resonant state or by the specific resonant state coupling is demonstrated with the one-dimensional complex potential energy curves of the temporary anion (cysteine)(-). The wave function of the lowest shape resonant state of the temporary anion (cystine)(-) distinctly shows the localized anti-bond (S-S)* character, implying that this disulfide bond can be easily broken due to the low-energy electron resonant attachment.     

Macrocyclic scaffold for the collagen triple helix

[structure: see text] Three strands of natural collagen are linked by covalent bonds prior to their folding into a triple helix. We report on a synthetic collagen in which the strands are pendent on a rigid macrocyclic scaffold of C(3) symmetry. The scaffold confers substantial conformational stability upon the collagen triple helix and makes its folding independent of concentration, both desirable attributes for exploring and exploiting synthetic collagens.     

Asteropine A, a sialidase-inhibiting conotoxin-like peptide from the marine sponge Asteropus simplex

Marine sponges contain structurally intriguing and biologically active peptides of nonribosomal peptide synthase origin, often containing amino acids with novel structures. Here we report the discovery of asteropine A (APA), a cystine knot to be isolated from marine sponges. The solution structure of APA as determined by NMR belongs to the four-loop class of cystine knots similar to those of some conotoxins and spider toxins. However, the highly negatively charged surface of APA is uncommon among other cystine knots. APA competitively inhibits bacterial sialidases, but not a viral sialidase. APA was inactive against all other enzymes tested and did not have any apparent antitumor activity. Our data suggest that APA and other knotting peptides may be important leads for antibacterial and even antiviral drug development.     

Effect of 3-hydroxyproline residues on collagen stability

Collagen is an integral part of many types of connective tissue in animals, especially skin, bones, cartilage, and basement membranes. A fibrous protein, collagen has a triple-helical structure, which is comprised of strands with a repeating Xaa-Yaa-Gly sequence. l-Proline (Pro) and 4(R)-hydroxy-l-proline (4-Hyp) residues occur most often in the Xaa and Yaa positions. The 4-Hyp residue is known to increase markedly the conformational stability of a collagen triple helix. In natural collagen, a 3(S)-hydroxy-l-proline (3-Hyp) residue occurs in the sequence: 3-Hyp-4-Hyp-Gly. Its effect on collagen stability is unknown. Here, two host-guest peptides containing 3-Hyp are synthesized: (Pro-4-Hyp-Gly)(3)-3-Hyp-4-Hyp-Gly-(Pro-4-Hyp-Gly)(3) (peptide 1) and (Pro-4-Hyp-Gly)(3)-Pro-3-Hyp-Gly-(Pro-4-Hyp-Gly)(3) (peptide 2). The 3-Hyp residues in these two peptides diminish triple-helical stability in comparison to Pro. This destabilization is small when 3-Hyp is in the natural Xaa position (peptide 1). There, the inductive effect of its 3-hydroxyl group diminishes slightly the strength of the interstrand 3-HypC=O.H-NGly hydrogen bond. The destabilization is large when 3-Hyp is in the nonnatural Yaa position (peptide 2). There, its pyrrolidine ring pucker leads to inappropriate mainchain dihedral angles and interstrand steric clashes. Thus, the natural regioisomeric residues 3-Hyp and 4-Hyp have distinct effects on the conformational stability of the collagen triple helix.     

Metal-assisted stabilization and probing of collagenous triple helices

Collagen model peptides that contain 2,2'-bipyridyl (bpy) ligands were designed and synthesized. The thermal stability of the collagenous triple helix was increased by forming an Fe(II)(bpy-peptide)(3) complex. The chirality of the metal center was shifted to form right-handed Delta-isomers induced by the supercoiling of the peptide moiety. Moreover, the refolding rate of the triple helix was increased in the presence of Fe(II). This metal-coordinating system possesses potential to be used to stabilize the triple-helical conformation as well as to probe the folding status of collagen model peptides.     

A hyperstable collagen mimic

BACKGROUND: Collagen is the most abundant protein in animals. Each polypeptide chain of collagen is composed of repeats of the sequence: Gly-X-Y, where X and Y are often L-proline (Pro) and 4(R)-hydroxy-L-proline (Hyp) residues, respectively. These chains are wound into tight triple helices of great stability. The hydroxyl group of Hyp residues contributes much to this conformational stability. The existing paradigm is that this stability arises from interstrand hydrogen bonds mediated by bridging water molecules. This model was tested using chemical synthesis to replace Hyp residues with 4(R)-fluoro-L-proline (Flp) residues. The fluorine atom in Flp residues does not form hydrogen bonds but does elicit strong inductive effects. RESULTS: Replacing the Hyp residues in collagen with Flp residues greatly increases triple-helical stability. The free energy contributed by the fluorine atom in Flp residues is twice that of the hydroxyl group in Hyp residues. The stability of the Flp-containing triple helix far exceeds that of any untemplated collagen mimic of similar size. CONCLUSIONS: Bridging water molecules contribute little to collagen stability. Rather, collagen stability relies on previously unappreciated inductive effects. Collagen mimics containing fluorine or other appropriate electron-withdrawing substituents could be the basis of new biomaterials for restorative therapies.     

The kinetics of helix unfolding of an azobenzene cross-linked peptide probed by nanosecond time-resolved optical rotatory dispersion

The unfolding dynamics of a 16 amino acid peptide (Ac-EACAREAAAREAACRQ-NH(2), FK-11-X) was followed using nanosecond time-resolved optical rotatory dispersion (ORD). The peptide was coupled to an azobenzene linker that undergoes subnanosecond photoisomerization and reisomerizes on a time scale of minutes. When the linker is in the trans form, the peptide favors a more helical structure (66% helix/34% disordered) and when in the cis configuration the helical content is reduced. Unfolding of FK-11-X was rapidly triggered by a 7-ns laser pulse at 355 nm, forming cis azobenzene-linked peptides that maintained the secondary structure (helical or disordered) of their trans azobenzene counterparts. The incompatibility of the instantaneous cis photoproduct with helical secondary structure drives the subsequent peptide unfolding to a new conformational equilibrium between cis helix and cis disordered structures. The kinetic results show a approximately 40% decrease in the time-dependent ORD signal at 230 nm that is best fit to a single-exponential decay with a time constant of 55 +/- 6 ns. Folding and unfolding rates for cis FK-11-X are estimated to be approximately 3.0 x 10(6) s(-)(1) (1/330 ns) and approximately 1.5 x 10(7) s(-)(1) (1/66 ns), respectively.     

Characterization of collagen model peptides containing 4-fluoroproline; (4(S)-fluoroproline-pro-gly)10 forms a triple helix,but (4(R)-fluoroproline-pro-gly)10 does not

Collagen model peptide (Pro-Pro-Gly)10 has a triple helical structure and undergoes a thermal transition to a single random coil structure. The transition temperature of the analogous model peptides depends largely on amino acid substitution. Substitution of Pro by 4-hydroxyproline (Hyp) or 4-fluoroproline (fPro) has especially attracted attention because the position of substitution and chirality of the hydroxyl group or fluorine atom affect the transition temperatures. Here, we demonstrated that (4(S)-fPro-Pro-Gly)10 takes a triple helical structure, but (4(R)-fPro-Pro-Gly)10 exists in a single chain structure. This is not consistent with the case of Hyp substitution in our previous report where both (4(S)-Hyp-Pro-Gly)10 and (4(R)-Hyp-Pro-Gly)10 are in a single random coil state.     

Double-helical cyclic peptides: design, synthesis, and crystal structure of figure-eight mirror-image conformers of adamantane-constrained cystine-containing cyclic peptide cyclo (Adm-Cyst)(3)

A large number of macrocycles containing alternating repeats of cystine diOMe(-NH-CH(CO(2)Me)-CH(2)-S-)(2) and either a conformationally rigid aromatic/alicyclic moiety or a flexible polymethylene unit (X) in the cyclic backbone with ring size varying from 13- to 78-membered have been examined by spectral ((1)H NMR, FT-IR, CD) and X-ray crystallography studies for unusual conformational preferences. While (1)H NMR measurements indicated a turnlike conformation for all macrocycles, stabilized by intramolecular NH.CO hydrogen bonding, as also supported by FT-IR spectra in chloroform, convincing proof for beta-turn structures was provided by circular dichroism studies. Single-crystal X-ray studies on 39-membered cyclo (Adm-L-Cyst)(3) revealed a double-helical fold (figure-eight motif) for the macrocycle. Only a right-handed double helix was seen in the macrocycle constructed from L-cystine. The mirror-image macrocycle made up of D-cystine units exhibited a double helix with exactly the opposite screw sense, as expected. The enantiomeric figure-eights were stabilized by two intramolecular NH. CO hydrogen bonds and exhibited identical (1) H NMR and FT-IR spectra. The CD spectra of both isomers had a mirror-image relationship. The present results have clearly brought out the importance of cystine residues in inducing turn conformation that may be an important deciding factor for the adoption of topologically important structures by macrocycles containing multiple S-S linkages.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric observation of a peptide triplet induced by thermal cleavage of cystine.

Heat-induced (90 degrees C, 30 min) beta-elimination of a cystine residue leads to cleavage of a disulfide bond and produces a set of three peptides with a cysteine residue, a thiocysteine residue (+32Da), and a dehydroalanine residue (-34Da). This characteristic feature was observed from somatostatin and insulin by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Mass spectrometric observation of this triplet is useful in identifying the presence of a cystine residue in a peptide, and could assist mass spectrometric identification of the peptide from a database.     

Positive and negative design leads to compositional control in AAB collagen heterotrimers

Although collagen is the most abundant protein in the human body and has at least 28 types, research involving collagen mimetic systems only recently began to consider the innate ability of collagen to control helix composition and register. Collagen triple helices can be homotrimeric or heterotrimeric, and while some types of natural collagen form only one specific composition of helix, others can form multiple compositions. It is critical to fully understand and, if possible, reproduce the control that native collagen has on helix composition and register. In this Article, we utilize both positive and negative design for the assembly of specific AAB heterotrimers using charged amino acids to form intrahelix electrostatic interactions, which promote heterotrimer formation and simultaneously discourage homotrimers. Homotrimers are further discouraged by reducing hydroxyproline content, which would otherwise lead to nonspecific promotion of triple helix formation. We combine peptides in a 2:1 ratio in which the more abundant peptide has a charge 1/2 and opposite of the less abundant peptide, which can result in the formation of a zwitterionically neutral AAB heterotrimer. Using this approach, we are able to design collagen mimetic systems with full control over the composition of the resulting triple helix. All previous reports on synthetic collagen heterotrimers have shown mixed populations with respect to composition due to varying amounts of residual homotrimers. Our results yield a greater understanding of the self-assembly of collagenous sequences as well as provide a novel design scheme, both positive and negative, for the synthesis of extracellular matrix mimetics.     

HPLC simultaneous analysis of thiols and disulfides: on-line reduction and indirect fluorescence detection without derivatization

A liquid chromatography method with indirect fluorescence detection has been developed for simultaneous detection of cysteine, cystine, homocysteine, homocystine, glutathione and glutathione disulfide. After separation in their native forms, a post-column solution of tris(2-carboxyethyl)phosphine (TCEP) at 32 mM, pH 8 is added to reduce the disulfides on-line to the corresponding thiol. The effluent is then merged with a second post-column solution of the highly fluorescent complex Cd(HQS)(2)(2-). The cadmium is complexed by the eluting thiols, effectively quenching the fluorescence. Optimization of the separation, the on-line reduction and the indirect fluorescence detection are discussed. Detection limits from 0.3-4.3 microM (0.04 to 2.6 ppm) are achieved for the six analytes in a 20 min separation.     

Computational design of a collagen A:B:C-type heterotrimer

We have successfully designed an A:B:C collagen peptide heterotrimer using an automated computational approach. The algorithm maximizes the energy gap between the target and competing misfolded states while enforcing a minimum target stability. Circular dichroism (CD) measurements confirm that all three peptides are required to form a stable, structured triple helix. This study highlights the power of automated computational design, providing model systems to probe the biophysics of collagen assembly and developing general methods for the design of fibrous proteins.