The role of cystine knots in collagen folding and stability,part I. Conformational properties of (Pro-Hyp-Gly)5 and (Pro-(4S)-FPro-Gly)5 model trimers with an artificial cystine knot

In analogy to the cystine knots present in natural collagens, a simplified disulfide cross-link was used to analyse the conformational effects of a C-terminal artificial cystine knot on the folding of collagenous peptides consisting of solely (Pro-Hyp-Gly) repeating units. Assembly of the alpha chains into a heterotrimer by previously applied regioselective disulfide-bridging strategies failed because of the high tendency of (Pro-Hyp-Gly)(5) peptides to self-associate and form homotrimers. Only when side-chain-protected peptides were used, for example in the Hyp(tBu) form, and a new protection scheme was adopted, selective interchain-disulfide cross-linking into the heterotrimer in organic solvents was successful. This unexpected strong effect of the conformational properties on the efficiency of well-established reactions was further supported by replacing the Hyp residues with (4S)-fluoroproline, which is known to destabilise triple-helical structures. With the related [Pro-(4S)-FPro-Gly](5) peptides, assembly of the heterotrimer in aqueous solution proceeded in a satisfactory manner. Both the intermediates and the final fluorinated heterotrimer are fully unfolded in aqueous solution even at 4 degrees C. Conversely, the disulfide-crossbridged (Pro-Hyp-Gly)(5) heterotrimer forms a very stable triple helix. The observation that thermal unfolding leads to scrambling of the disulfide bridges was unexpected. Although NMR experiments support an extension of the triple helix into the cystine knot, thermolysis is not associated with the unfolding process. In fact, the unstructured fluorinated trimer undergoes an equally facile thermodegradation associated with the intrinsic tendency of unsymmetrical disulfides to disproportionate into symmetrical disulfides under favourable conditions. The experimental results obtained with the model peptides fully support the role of triple-helix nucleation and stabilisation by the artificial cystine knot as previously suggested for the natural cystine knots in collagens.     

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.     

New, simple method for synthesis of cystine peptides

A report is given on the conversion of S-trityl-cysteine-containing protected peptides to cystine peptides by a reaction with iodine in methanol. The new method permits the synthesis of symmetrical, open-chain unsymmetrical, and especially cyclic cystine peptides.     

Synthesis of Chiral Tetrahomodiazacalix[4]-and [6]Arenes Bridged by Cystine Peptide

Bicyclic tetrahomodiazacalix[4]- and [6]arenes bridged by a cystine peptide were easily synthesized from the cyclization reactions of bis(chloromethyl)phenol-formaldehyde oligomers with cystine peptides in DMF in moderate yields.     

The Synthesis of Cystine Peptides by Iodine Oxidation of S-Trityl-cysteine and S-Acetamidomethyl-cysteine Peptides

Previously reported studies of the iodine oxidation of S-trityl-cysteine peptides and S-acetamidomethyl-cysteine peptides, leading directly to cystine peptides, have been extended. Detailed investigations have been made of the reactivities of the S-trityl and the S-acetamidomethyl group towards iodine in various solvents. In chloroform, methylene chloride, trifluoroethanol, and hexafluoroisopropyl alcohol the differences in the reaction rates of the two groups have been found to be extremely large, allowing the selective conversion of the tritylthio groups to disulfides in the presence of the S-acetamidomethyl derivatives. In a second group of solvents, consisting of methanol, acetic acid, dioxane, and mixtures of these solvents with water, simultaneous iodine oxidation of S-trityl- and S-acetamidomethyl-cysteine peptides leads to a preferential combination of these two residues, resulting in predominantly asymmetrical cystine derivatives. - The suitability of the two sulfur-protecting groups in the synthesis of cyclic cystine peptides has been assessed. - Possible reaction mechanisms are discussed. - The scope and limitations of iodine oxidation in peptide synthesis have been studied. The applicability of the method has been demonstrated in the preparation of the open-chain asymmetrical cystine peptide 5 , the protected somatostatin derivative 17 , and the A(1–13) segment 19 of human insulin, previously employed in the total synthesis of this hormone.     

A novel approach to the regioselective synthesis of a disulfide-linked heterodimeric bicyclic peptide mimetic of brain-derived neurotrophic factor

We describe a novel acetamidomethyl to S-pyridinyl exchange that is used for the synthesis of a multi-disulfide-linked and constrained heterodimeric bicyclic peptide mimetic of brain-derived neurotrophic factor (BDNF). This simple and effective method should be readily transferable to the synthesis of similar disulfide-linked heterodimeric peptides, as well as being of general utility for the synthesis of peptides bearing multiple cystine frameworks.     

Quantitative assessment of cysteine and cystine in peptides and proteins following organomercurial derivatization and analysis by matrix-assisted laser desorption ionization mass spectrometry

Protocols for the analysis of the sulfhydryl content in peptides and proteins using chemical derivatization by organomercurial reagents and analysis by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) have been developed. The number of reactive cysteine residues in peptides and proteins can be determined by exploiting the affinity and selectivity of organomercurial reagents for macromolecular thiols. Mass shifts observed in MALDI mass spectra obtained before and after cysteine derivatization with p-hydroxy-mercuribenzoate (pHMB) permit the number of free sulfhydryl groups to be determined. The pHMB derivative of each free cysteine residue provides a mass shift of 321 u, overcoming limitations in the mass resolution of MALDI time-of-flight mass spectrometry. Reactive cysteine residues in a macromolecule can be selectively derivatized by using a fivefold molar excess of pHMB reagent. Total sulfhydryl content (i.e., cysteine and cystine) can be determined after disulfide reduction. However, analyses for total cysteine content are more complex, requiring protein denaturation, cystine reduction, and sample purification before derivatization and analysis by MALDI-MS. Conditions for sample denaturation, alkyl-phosphine reduction, pHMB derivatization, and sample purification by analyte adsorption and desalting on protein transfer membranes, are described for cysteine/cystine analysis performed on microgram (10–200 pmol) quantities of somatostatin, insulin, hemoglobin, and β-lactoglobulin.     

Facile formation of chiral calixarene analogs incorporating cystine peptide into the macrocyclic ring

Chiral calixarene analogs incorporating cystine peptide into their macrocyclic ring were easily prepared by the cyclization reactions of bis(chloromethyl)phenol-formaldehyde oligomers with cystine peptides in moderate yields. Circular dichroism (CD) spectra indicated the existence of the transmission of the chirality from peptide unit to phenol-formaldehyde oligomer moiety.     

Prediction of Antibacterial Peptides against Propionibacterium acnes from the Peptidomes of Achatina fulica Mucus Fractions

Acne vulgaris is a common skin disease mainly caused by the Gram-positive pathogenic bacterium, Propionibacterium acnes. This bacterium stimulates the inflammation process in human sebaceous glands. The giant African snail (Achatina fulica) is an alien species that rapidly reproduces and seriously damages agricultural products in Thailand. There were several research reports on the medical and pharmaceutical benefits of these snail mucus peptides and proteins. This study aimed to in silico predict multifunctional bioactive peptides from A. fulica mucus peptidome using bioinformatic tools for the determination of antimicrobial (iAMPpred), anti-biofilm (dPABBs), cytotoxic (ToxinPred) and cell-membrane-penetrating (CPPpred) peptides. Three candidate peptides with the highest predictive score were selected and re-designed/modified to improve the required activities. Structural and physicochemical properties of six anti-P. acnes (APA) peptide candidates were performed using the PEP–FOLD3 program and the four previous tools. All candidates had a random coiled structure and were named APAP-1 ori, APAP-2 ori, APAP-3 ori, APAP-1 mod, APAP-2 mod, and APAP-3 mod. To validate the APA activity, these peptide candidates were synthesized and tested against six isolates of P. acnes. The modified APA peptides showed high APA activity on three isolates. Therefore, our biomimetic mucus peptides could be useful for preventing acne vulgaris and further examined on other activities important to medical and pharmaceutical applications.     

Reverse phase high pressure liquid chromatography for the separation of peptide hormone diastereoisomers

Diastereoisomers of specifically labeled oxytocin derivatives were resolved using reverse phase high pressure liquid chromatography. The peptides [1-hemi-DL-[alpha-2H]cystine]oxytocin, [6-hemi-DL-[alpha-2H]cystine]oxytocin, [2-DL-[alpha-2H]tyrosine]oxytocin and[8-DL-[2-13C]leucine]oxytocin were readily separated using the conditions described. The diastereoisomers of the oxytocin analog [3-DL-[2-13C]leucine]oxytocin also demonstrated baseline resolution under the same conditions. The procedure offers the investigator a rapid method for screening synthetic oxytocin peptides for undesirable diastereoisomeric by-products.     

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.      

Diaminodiacid Bridges to Improve Folding and Tune the Bioactivity of Disulfide‐Rich Peptides

Disulfide‐rich peptides containing three or more disulfide bonds are promising therapeutic and diagnostic agents, but their preparation is often limited by the tedious and low‐yielding folding process. We found that a single cystine‐to‐diaminodiacid replacement could significantly increase the folding efficiency of disulfide‐rich peptides and thus improve their production yields. The practicality of this strategy was demonstrated by the synthesis and folding of derivatives of the μ‐conotoxin SIIIA, the preclinical hormone hepcidin, and the trypsin inhibitor EETI‐II. NMR and X‐ray crystallography studies confirmed that these derivatives of disulfide‐rich peptide retained the correct three‐dimensional conformations. Moreover, the cystine‐to‐diaminodiacid replacement enabled structural tuning, thereby leading to an EETI‐II derivative with higher bioactivity than the native peptide.     

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.     

Dendronized molecular knots: selective synthesis of various generations,enantiomer separation,circular dichroism

For the first time, knot molecules (of the amide type) are synthesized, which bear one to three dendritic units of various generations at their periphery. They were obtained through two different routes: i) attachment of dendritic wedges to new mono-, di- and trihydroxy functionalized dodecaamide knots that have been obtained by selective debenzylation of oligobenzyloxy substituted knots, or ii) cyclization of already dendron substituted pyridine-2,6-dicarbonyl dichlorides with an "extended diamine" to directly yield the "tri-dendroknots". The derivatization of knot molecules by functional substituents and even large dendritic units is an important advance in the synthesis and property variation of molecular knots. This holds true in particular for substitution of the pyridine units of the knots, whereas the isophthalic acid units seem not to tolerate larger substitutents, as reflected in lower knot yields. These syntheses also demonstrate knots to be accessible indirectly by substitution of the corresponding mono-, di- and tri-functionalized knot skeleton. An advantage of dendritic "decoration" is the control of solubility and chromatographic behaviour of the molecular knots (knotanes). Suggestions are made about the threading mechanism by supramolecular template effects leading to the formation of amide-based molecular knots. The topological chirality of the new "dendroknots" is shown by efficient enantioseparations (separation factor alpha between 1.22 and 1.48). For this purpose (commercially unavailable) chiral column material of the Chiralpak type was used, in which the chiral component is covalently bonded to the silica gel support. The racemate splittings provide additional evidence for the knotted structure, as all other conceivable isomers such as macromonocyclic or catenated dodecaamides would not be chiral. The pure enantiomers obtained exhibit pronounced Cotton effects in their circular dichroism spectra. By comparison with the unsubstituted knot, the absolute configuration (Lambda, Delta) of all new knots is derived.     

On the relation between polypeptide structure and spredptogenic activity. 1. Peptides of cystine

The authors propose for the characterization of strepogenic substances of known composition the specific activity, i. e. the number of WOOLLEY units per μmole. Starting from L -leucyl-L -cystinyl-L -leucyl-L -valyl-L -glutamic acid (of very high strepogenine activity, 400 WOOLLEY units per mg, 230 WOOLLEY units per μmole) seven peptides have been synthesized by suppression and/or replacement of amino acid residues. A study of the relation between activity and structure of these peptides shows that:

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2‐Nitroveratryl as a Photocleavable Thiol‐Protecting Group for Directed Disulfide Bond Formation in the Chemical Synthesis of Insulin

Chemical synthesis of peptides can allow the option of sequential formation of multiple cysteines through exploitation of judiciously chosen regioselective thiol‐protecting groups. We report the use of 2‐nitroveratryl (oNv) as a new orthogonal group that can be cleaved by photolysis under ambient conditions. In combination with complementary S‐pyridinesulfenyl activation, disulfide bonds are formed rapidly in situ. The preparation of Fmoc‐Cys(oNv)‐OH is described together with its use for the solid‐phase synthesis of complex cystine‐rich peptides, such as insulin.     

Protein knots and fold complexity: some new twists

The current knowledge on topological knots in protein structure is reviewed, considering in turn, knots with three, four and five strand crossings. The latter is the most recent to be identified and has two distinct topological forms. The knot observed in the protein structure is the form that requires the least number of strand crossings to become un-knotted. The position of the chain termini must also correspond to a position that allows (un) knotting in one move. This is postulated as a general property of protein knots and other more complex knots with this property are proposed as the next most likely knots that might be found in a protein. It is also noted that the "Jelly-roll" fold found in some all-beta proteins would provide likely candidates. Alternative measures of knottedness and entanglement are reviewed, including the occurrence of slip-knots. These measures are related to the complexity of the protein fold and may provide useful filters for selecting predicted model structures.     

Syntheses and properties of dithia‐tetrahomodiaza‐calix[4]arenes bridged by cystine unit

Dithia‐tetrahomodiaza‐calix[4]arenes were synthesized by the cyclization reactions of bis(3‐(chloro‐methyl)‐2‐hydroxyphenyl)sulfide with cystine peptides in moderate yields. Conformational analysis of the macrocycles by using nmr spectroscopy reveled that the cyclophanes adopt a cone‐like form as a preferable conformation and the cystine bridge moiety is incorporated in the cavity. The calixarene analogs can extract transion metals such as Zn²⁺ and Cu²⁺ ions from an aqueous phase into chloroform.