Synthesis and in vitro hydroxyapatite binding of peptides conjugated to calcium-binding moieties

To confer bone-binding properties to proteins and other biological agents that lack specific targeting capacity, model peptide-based molecules were synthesized containing poly(aspartic acid), poly(glutamic acid), or a bisphosphonate (pamidronate). These motifs have well-documented affinities to hydroxyapatite, a property desirable for the targeting of molecules to bone for drug delivery and tissue engineering applications. Model peptides of increasing molecular mass (5-33 amino acids) were directly conjugated to eight aspartic acids (Asp8), eight glutamic acids (Glu8), or pamidronate, purified by high-performance liquid chromatography, and characterized by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy. The modified peptides were incubated with hydroxyapatite in phosphate-buffered saline at physiological conditions over 24 h. This study revealed a significant amount (>90%) of conjugated peptides adsorbed to the hydroxyapatite as compared to unmodified peptides (<5%). It was found that while there were significant differences between the different hydroxyapatite-binding and control groups for all time points, the size of the peptide had no statistical effect on peptide-hydroxyapatite binding. These results demonstrate that bisphosphonate and oligopeptide conjugates hold great promise for the development of new bioactive molecules for bone-specific applications.     

Effect of osteonectin-derived peptide on the viscoelasticity of hydrogel/apatite nanocomposite scaffolds

Hydrogel/apatite nanocomposites are the ideal biomaterial to mimic the physio-chemical and biologic properties of the bone and to fabricate scaffolds for bone regeneration. The objective of this work was to investigate the effect of an osteonectin derived glutamic acid sequence on the viscoelastic properties of poly(lactide-ethylene oxide-fumarate) (PLEOF)/apatite composite, as a model degradable material in bone regeneration. Osteonectin is an extracellular acidic glycoprotein of the bone matrix, which is believed to be involved in linking the collagen network to hydroxyapatite (HA), the mineral phase of the bone. We synthesized a 6-glutamic acid sequence in solid phase with affinity to HA crystals via ionic interactions. One end of the synthesized peptide was functionalized with an acrylate group to covalently attach the peptide (Ac-Glu6) to the aqueous-based biodegradable and in situ crosslinkable PLEOF hydrogel matrix. To determine the effect of energetic interactions between the fillers and hydrogel matrix, HA nanoparticles were also treated with an acrylate functionalized 6-glycine amino acid peptide (Ac-Gly6) that interacts with the fillers only by van der Waals and polar interactions (without ionic interactions). Crosslinked PLEOF/apatite scaffolds were prepared using PLEOF as the degradable macromer, HA nanofillers treated with Ac-Glu6 peptide linker, and a neutral redox initiation system. The viscoelastic properties were studied by dynamic time sweep, strain sweep, and small amplitude oscillatory rheometry. Composites without surface treatment, treated with Ac-Gly6, and treated with Ac-Glu6 at different volume fractions and various particle sizes were examined. The results showed that the 6-mer glutamic acid sequence significantly affects the shear modulus of the scaffold because of ionic interactions between the peptide and HA crystals.     

Multimerization of the Drosophila zeste protein is required for efficient DNA binding.

The Drosophila zeste protein forms multimeric species in vitro through its C-terminal domain. Multimerization is required for efficient binding to DNA containing multiple recognition sequences and increasing the number of binding sites stimulates binding in a cooperative manner. Mutants that can only form dimers still bind to a dimeric site, but with lower affinity. Mutations or progressive deletions from the C-terminal show that when even dimer formation is prevented, DNA-binding activity is lost. Surprisingly, binding activity is regained with larger deletions that leave only the DNA-binding domain. Additional protein sequences apparently inhibit DNA binding unless they permit multimerization. The DNA-binding domain peptides bind strongly even to isolated recognition sequences and they bind as monomers. The ability of various zeste peptides to stimulate white gene expression in vivo shows that multimeric forms are the functional species of the zeste product in vivo. The DNA-binding domain peptide binds well to DNA in vitro, but it cannot stimulate white gene expression in vivo. This failure may reflect the need for an activation domain or it may be caused by indiscriminate binding of this peptide to non-functional isolated sites. Multimerization increases binding specificity, selecting only sites with multiple recognition sequences.     

Selection and Finding of Lead Peptides Dual-targeting MMP14 and Metal Ions by Subtractive Cell Surface Panning and Molecular Docking

Subtractive cell surface panning from phage random peptide library and molecular docking were used to select and find binding peptides dual-targeting MMP14 and metal ions on MG-63 cells. Phages were abundantly enriched and totally 22 different peptide sequences were obtained. We demonstrated that the affinity phage peptides were not only targeted to MMP14 but also had affinities for zinc and nickel ions. Four possible peptide consensus sequences were identified as: AHQ/_SLH/_P, L/_I/_EPLL/_I, T/Q/_DARH/_FQ, MK/_PSR. The representative peptides were found to dock well to MMP14 at the sites around aa. 120–125 which were also the catalytic region of zinc binding sites on MMP14 by MVD molecular docking. The selected monoclonal phages could efficiently bind to the MMP14 expressed MG-63 cells and this again confirmed the targeting binding of the peptides to MMP14. In vitro study revealed that the selected monoclonal phages could greatly inhibit the viability of MG-63 cells. Also, the four synthetic representative consensus peptides inhibited the viability of MG-63 and HepG2 cells at μg levels. The consensus sequences identified in our study were newly reported and relatively specific to MMP14 and this may be further used for MMP14 based peptide drug development and/or diagnostic analyses.     

双靶向MMP-14和金属离子的结合肽筛选与分子模拟

以基质金属蛋白酶-14(MMP-14)催化结构域为靶标,通过噬菌体随机十二肽库 筛选和分子模拟、细胞免疫荧光、金属离子亲和层析以及体外细胞作用测定等技 术,进行了双靶向MMP-14和金属离子小分子结合多肽的筛选与研究.经4轮筛选, 噬菌体得到有效富集并获得13条不同的多肽序列.序列分析显示,可能的一致序列 有：AHQLH、HHXH、EI/LPLL/I.分子模拟与对接进一步确认一致序列AHQLH、HHTH 、LPLL与MMP-14催化结构域的氨基酸120~125区域良好分子对接并具有一定的专 一性,多条MMP-14结合肽不仅靶向MMP-14,同时结合金属离子.细胞生物学研究确 认,所测定的结合肽噬菌体对MMP-14诱导表达的MG63细胞具有良好的结合作用,揭 示结合肽对MMP-14的靶向结合特性,并且合成的AHQLH、LPLL一致序列多肽对MG63 细胞活力具有一定的抑制能力.这些新的和具有一定MMP-14专一性的一致序列可望 用于靶向MMP-14抗肿瘤药物的研发和利用.     

Interaction of calcium ions and salivary acidic proline-rich proteins with hydroxyapatite. A possible aspect of inhibition of hydroxyapatite formation.

The relationship between Ca2+- and hydroxyapatite-binding sites in salivary acidic proline-rich phosphoproteins A and C was investigated. Coating of hydroxyapatite with protein before adsorption had no effect on Ca2+ binding to the mineral, but simultaneous adsorption of Ca+ and protein to hydroxyapatite caused additional Ca2+ binding to the solid. The additional amount of Ca2+ adsorbed, measured in mol of Ca2+/mol of protein adsorbed to hydroxyapatite, was approx. 2 for protein C, 4 for protein A, 9 for the N-terminal tryptic peptide and 2 for dephosphorylated protein A. It is suggested that the ability of the proteins to inhibit hydroxyapatite formation is related to the binding of the proteins to crystal growth sites on the mineral, which prevents access of Ca2+ from the surrounding liquid.     

Epitope motif of an anti‐nitrotyrosine antibody specific for tyrosine‐nitrated peptides revealed by a combination of affinity approaches and mass spectrometry

Nitration of tyrosine residues has been shown to be an important oxidative modification in proteins and has been suggested to play a role in several diseases such as atherosclerosis, asthma, lung and neurodegenerative diseases. Detection of nitrated proteins has been mainly based on the use of nitrotyrosine‐specific antibodies. In contrast, only a small number of nitration sites in proteins have been unequivocally identified by MS. We have used a monoclonal 3‐NT‐specific antibody, and have synthesized a series of tyrosine‐nitrated peptides of prostacyclin synthase (PCS) in which a single specific nitration site at Tyr‐430 had been previously identified upon reaction with peroxynitrite 17 . The determination of antibody‐binding affinity and specificity of PCS peptides nitrated at different tyrosine residues (Tyr‐430, Tyr‐421, Tyr‐83) and sequence mutations around the nitration sites provided the identification of an epitope motif containing positively charged amino acids (Lys and/or Arg) N‐terminal to the nitration site. The highest affinity to the anti‐3NT‐antibody was found for the PCS peptide comprising the Tyr‐430 nitration site with a K_D of 60 nM determined for the peptide, PCS(424‐436‐Tyr‐430NO₂); in contrast, PCS peptides nitrated at Tyr‐421 and Tyr‐83 had substantially lower affinity. ELISA, SAW bioaffinity, proteolytic digestion of antibody‐bound peptides and affinity‐MS analysis revealed highest affinity to the antibody for tyrosine‐nitrated peptides that contained positively charged amino acids in the N‐terminal sequence to the nitration site. Remarkably, similar N‐terminal sequences of tyrosine‐nitration sites have been recently identified in nitrated physiological proteins, such as eosinophil peroxidase and eosinophil‐cationic protein. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

P. falciparum pro-histoaspartic protease (proHAP) protein peptides bind specifically to erythrocytes and inhibit the invasion process in vitro

Plasmodium falciparum histoaspartic protease (HAP) is an active enzyme involved in haemoglobin degradation. HAP is expressed as an inactive 51-kDa zymogen and is cleaved into an active 37-kDa enzyme. It has been proposed that this kind of protease might be implicated in the parasite's invasion of erythrocytes; however, this protein's role during invasion has still to be determined. Synthetic peptides derived from the HAP precursor (proHAP) were tested in erythrocyte binding assays to identify their possible function in the invasion process. Two proHAP high-activity binding peptides (HABPs) specifically bound to erythrocytes; these peptides were numbered 30609 (101LKNYIKESVKLFNKGLTKKS120) and 30610 (121YLGSEFDNVELKDLANVLSF140 ). The binding of these two peptides was saturable, presenting nanomolar affinity constants. These peptides interacted with 26- and 45-kDa proteins on the erythrocyte surface; the nature of these receptor sites was studied in peptide binding assays using enzyme-treated erythrocytes. The HABPs showed greater than 90% merozoite invasion inhibition in in vitro assays. Goat serum containing proHAP polymeric peptide antibodies inhibited parasite invasion in vitro .     

Preferential binding of apolipoprotein E derived peptides with oxidized phospholipid

The physiological function of apolipoprotein E (apoE) includes transport and metabolism of lipids and its C-terminal domain harbors high affinity lipid-binding sites. Although the binding of apoE with non-oxidized phospholipid containing membranes has been characterized earlier, the interaction of apoE or its fragments with oxidized phospholipid containing membrane has never been studied. In this study we have compared the interaction of amphipathic helical peptide sequences derived from the C-terminal domain of apoE with membrane vesicles containing oxidized phospholipid, 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), with membrane vesicles without PazePC. The interaction was studied by monitoring (a) fluorescence emission maxima of the peptides, (b) acrylamide quenching of the peptides tryptophan residues and (c) by measuring the equilibrium binding constants by resonance energy transfer (RET) analysis. Our result shows that peptide sequence 202-223, 245-266 and 268-289 of apoE has higher affinity towards membrane containing PazePC, compared to membrane without PazePC. Presence of 1mM divalent cation or 50 mM NaCl in the buffer decreased the binding of peptides to PazePC containing membrane vesicles suggesting possible involvement of the electrostatic interaction in the binding. These observations suggest that the preferential binding of apoE to oxidized phospholipid containing membrane may play a role in the anti-oxidative properties of apoE.     

Identification of model peptides as affinity ligands for the purification of humanized monoclonal antibodies by means of phage display.

A proof-of-principle study was initiated to determine whether phage-display technology could be used to identify peptides as leads in the customization of ligands for affinity chromatography and to identify a peptide or peptidomimetic for use as a Protein A alternative in the affinity purification of monoclonal antibodies. The constant region of humanized anti-Tac (HAT), prepared by pepsin digestion and receptor-affinity chromatography, was used as the target for phage display in this study. As such, 20 phage-derived peptide sequences were identified from four rounds of biopanning with two linear phage-display libraries (7-mer, containing 100 copies of 2 x 10(9) sequences and 12-mer, containing 70 copies of 1.4 x 10(9) sequences). Five peptides were synthesized for use as affinity ligands, based on sequence homology to Protein A, sequence redundancy, and amino acid motifs. The best HAT binding immobilized peptide was EPIHRSTLTALL. The best-fit analysis of this peptide sequence with Protein A yielded an alignment well within the Fc binding domain of Protein A. These results suggest that phage display can serve as a tool in the identification of peptides as model ligands for affinity chromatography.     

Effects of charge and hydrophobicity on the oligomerization of peptides derived from IAPP

Changes in pH resulting in modifications of charge can dramatically alter the folding and interaction of proteins. This article probes the effects of charge and hydrophobicity on the oligomerization of macrocyclic β-sheet peptides derived from residues 11–17 of IAPP (RLANFLV). Previous studies have shown that a macrocyclic β-sheet peptide containing this IAPP sequence (peptide 1_Arg) does not form oligomers in aqueous solution at low millimolar concentrations. Replacing arginine with the uncharged isostere citrulline generates a homologue (peptide 1_Cit) that forms a tetramer consisting of a sandwich of hydrogen-bonded dimers. The current study probes the role of charge and hydrophobicity by changing residue 11 to glutamic acid (peptide 1_Glu) and leucine (peptide 1_Leu). Diffusion-ordered spectroscopy (DOSY) studies show that peptides 1_Glu and 1_Leu form tetramers in solution. NOESY studies confirm that both peptides form the same sandwich-like tetramer as peptide 1_Cit. ¹H NMR spectroscopy at various concentrations reveals that peptide 1_Leu has the highest propensity to form tetramers. The effects of pH and charge on oligomerization are further probed by incorporating histidine at position 11 (peptide 1_His). DOSY studies show that peptide 1_His forms a tetramer at high pH. At low pH, peptide 1_His forms a new species that has not been previously observed by our research group—a dimer. These studies demonstrate the importance of charge and hydrophobicity in the oligomerization of IAPP-derived peptides.     

Bone acidic glycoprotein-75 delineates the extracellular sites of future bone sialoprotein accumulation and apatite nucleation in osteoblastic cultures

Addition of an organophosphate source to UMR osteoblastic cultures activates a mineralization program in which BSP localizes to extracellular matrix sites where hydroxyapatite crystals are subsequently nucleated. This study identifies for the first time novel extracellular spherical structures, termed biomineralization foci (BMF), containing bone acidic glycoprotein-75 (BAG-75), bone sialoprotein (BSP), and alkaline phosphatase that are the exclusive sites of initial nucleation of hydroxyapatite crystals in the UMR model. Importantly, in the absence of added phosphate, UMR cultures after reaching confluency contain two size populations of morphologically identifiable BMF precursors enriched in BAG-75 (15-25 and 150-250 microm in diameter). The shape and size of the smaller population are similar to structures assembled in vitro through self-association of purified BAG-75 protein. After organophosphate addition, BSP accumulates within these BAG-75-containing BMF precursors, with hydroxyapatite crystal nucleation occurring subsequently. In summary, BAG-75 is the earliest detectable biomarker that accurately predicts the extracellular sites of de novo biomineralization in UMR cultures. We hypothesize that BAG-75 may perform a key structural role in the assembly of BMF precursors and the recruitment of other proteins such as alkaline phosphatase and BSP. Furthermore, we propose a hypothetical mechanism in which BAG-75 and BSP function actively in nucleation of apatite within BMF.     

A novel integrin specificity exemplified by binding of the alpha v beta 5 integrin to the basic domain of the HIV Tat protein and vitronectin

Several studies have addressed the interaction of the HIV Tat protein with the cell surface. Our analysis of the cell attachment-promoting activity of Tat and peptides derived from it revealed that the basic domain of Tat, not the arg-gly-asp (RGD) sequence, is required for cell attachment to Tat. Affinity chromatography with Tat peptides and immunoprecipitation with various anti-integrin antibodies suggest that the vitronectin-binding integrin, alpha v beta 5, is the cell surface protein that binds to the basic domain of Tat. The Tat basic domain contains the sequence RKKRRQRRR. A related sequence, KKQRFRHRNRKG, present in the heparin-binding domain of an alpha v beta 5 ligand, vitronectin, also bound alpha v beta 5 in affinity chromatography and, in combination with an RGD peptide, was an inhibitor of cell attachment to vitronectin. The alpha v beta 5 interaction with these peptides was not solely due to high content of basic amino acids in the ligand sequences; alpha v beta 5 did not bind substantially to peptides consisting entirely of arginine or lysine, whereas a beta 1 integrin did bind to these peptides. The interaction of alpha v beta 5 with Tat is atypical for integrins in that the binding to Tat is divalent cation independent, whereas the binding of the same integrin to an RGD- containing peptide or to vitronectin requires divalent cations. These data define an auxiliary integrin binding specificity for basic amino acid sequences. These basic domain binding sites may function synergistically with the binding sites that recognize RGD or equivalent sequences.     

Biosynthesis of peptide neurotransmitters: studies on the formation of peptide amides

A high proportion of peptide transmitters and peptide hormones terminate their peptide chain in a C-terminal amide group which is essential for their biological activity. The specificity of an enzyme that catalyses the formation of the amide was investigated with the aid of synthetic peptide substrates. With peptides containing l-amino acids the enzyme exhibited an essential requirement for glycine in the C-terminal position; amidation did not take place with peptides that had leucine, alanine, glutamic acid, lysine or N-methylglycine at the C-terminus and a peptide extended by the attachment of lysine to the C-terminal glycine did not act as a substrate. Amidation did occur with a peptide containing C-terminal D-alanine but no reaction was detected with peptides having C-terminal, D-serine or D-leucine. In tripeptides with a neutral amino acid in the penultimate position, amidation, took place readily but the reaction was slower when this position was occupied by an acidic or a basic residue. A series of overlapping peptides with C-terminal glycine, based on partial sequences of calcitonin, underwent amidation at similar rates, indicating that the amidating enzyme recognizes only a limited sequence at the C-terminus of its substrates. The results provide evidence that the amidating enzyme has a highly compact substrate binding site.     

Cellular and viral peptides bind multiple sites on the N‐terminal domain of clathrin

Short peptide motifs in unstructured regions of clathrin‐adaptor proteins recruit clathrin to membranes to facilitate post‐Golgi membrane transport. Three consensus clathrin‐binding peptide sequences have been identified and structural studies show that each binds distinct sites on the clathrin heavy chain N‐terminal domain (NTD). A fourth binding site for adaptors on NTD has been functionally identified but not structurally characterised. We have solved high resolution structures of NTD bound to peptide motifs from the cellular clathrin adaptors β2 adaptin and amphiphysin plus a putative viral clathrin adaptor, hepatitis D virus large antigen (HDAg‐L). Surprisingly, with each peptide we observe simultaneous peptide binding at multiple sites on NTD and viral peptides binding to the same sites as cellular peptides. Peptides containing clathrin‐box motifs (CBMs) with the consensus sequence LΦxΦ[DE] bind at the ‘arrestin box’ on NTD, between β‐propeller blades 4 and 5, which had previously been thought to bind a distinct consensus sequence. Further, we structurally define the fourth peptide binding site on NTD, which we term the Royle box. In vitro binding assays show that clathrin is more readily captured by cellular CBMs than by HDAg‐L, and site‐directed mutagenesis confirms that multiple binding sites on NTD contribute to efficient capture by CBM peptides.      

Ligand regulates epidermal growth factor receptor kinase specificity: activation increases preference for GAB1 and SHC versus autophosphorylation sites

The epidermal growth factor receptor (EGFR) kinase catalyzes phosphorylation of tyrosines in its C terminus and in other cellular targets upon epidermal growth factor (EGF) stimulation. Here, by using peptides derived from EGFR autophosphorylation sites and cellular substrates, we tested the hypothesis that ligand may function to regulate EGFR kinase specificity by modulating the binding affinity of peptide sequences to the active site. Measurement of the steady-state kinetic parameters, K(m) and k(cat), revealed that EGF did not affect the binding of EGFR peptides but increased the binding affinity for peptides corresponding to the major EGFR-mediated phosphorylation sites of the adaptor proteins Gab1 (Tyr-627) and Shc (Tyr-317), and for peptides containing the previously identified optimal EGFR kinase substrate sequence EEEEYFELV (3-7-fold). Conversely, EGF stimulation increased k(cat) approximately 5-fold for all peptides. Thus, ligand changed the relative preference of the EGFR kinase for substrates as evidenced by EGF increases of approximately 5-fold in the specificity constants (k(cat)/K(m)) for EGFR peptides, whereas approximately 15-40-fold increases were observed for other peptides, such as Gab1 Tyr-627. Furthermore, we demonstrate that EGF (i) increased the binding affinity of EGFR to Gab1 Tyr-627 and Shc Tyr-317 sites in purified GST fusion proteins approximately 4-6-fold, and (ii) EGF significantly enhanced the phosphorylation of these sites, relative to EGFR autophosphorylation, in cell lysates containing the full-length Gab1 and Shc proteins. Analysis of peptides containing amino acid substitutions indicated that residues C-terminal to the target tyrosine were critical for EGF-stimulated increases in substrate binding and regulation of kinase specificity. To our knowledge, this represents the first demonstration that ligand can alter specificity of a receptor kinase toward physiologically relevant targets.     

PBMC activation via the ERK and STAT signaling pathways enhances the anti-tumor activity of <Emphasis Type="Italic">Staphylococcal</Emphasis> enterotoxin A

In a recent study, we highlighted the importance of cationic charge and arginine residues for the neuroprotective properties of poly-arginine and arginine-rich peptides. In this study, using cortical neuronal cultures and an in vitro glutamic acid excitotoxicity model, we examined the neuroprotective efficacy of different modifications to the poly-arginine-9 peptide (R9). We compared an unmodified R9 peptide with R9 peptides containing the following modifications: (i) C-terminal amidation (R9-NH2); (ii) N-terminal acetylation (Ac-R9); (iii) C-terminal amidation with N-terminal acetylation (Ac-R9-NH2); and (iv) C-terminal amidation with d-amino acids (R9D-NH2). The three C-terminal amidated peptides (R9-NH2, Ac-R9-NH2, and R9D-NH2) displayed neuroprotective effects greater than the unmodified R9 peptide, while the N-terminal acetylated peptide (Ac-R9) had reduced efficacy. Using the R9-NH2 peptide, neuroprotection could be induced with a 10 min peptide pre-treatment, 1–6 h before glutamic acid insult, or when added to neuronal cultures up to 45 min post-insult. In addition, all peptides were capable of reducing glutamic acid-mediated neuronal intracellular calcium influx, in a manner that reflected their neuroprotective efficacy. This study further highlights the neuroprotective properties of poly-arginine peptides and provides insight into peptide modifications that affect efficacy.     

Peptide array‐based characterization and design of ZnO‐high affinity peptides

Peptides with both an affinity for ZnO and the ability to generate ZnO nanoparticles have attracted attention for the self‐assembly and templating of nanoscale building blocks under ambient conditions with compositional uniformity. In this study, we have analyzed the specific binding sites of the ZnO‐binding peptide, EAHVMHKVAPRP, which was identified using a phage display peptide library. The peptide binding assay against ZnO nanoparticles was performed using peptides synthesized on a cellulose membrane using the spot method. Using randomized rotation of amino acids in the ZnO‐binding peptide, 125 spot‐synthesized peptides were assayed. The peptide binding activity against ZnO nanoparticles varied greatly. This indicates that ZnO binding does not depend on total hydrophobicity or other physical parameters of these peptides, but rather that ZnO recognizes the specific amino acid alignment of these peptides. In addition, several peptides were found to show higher binding ability compared with that of the original peptides. Identification of important binding sites in the EAHVMHKVAPRP peptide was investigated by shortened, stepwise sequence from both termini. Interestingly, two ZnO‐binding sites were found as 6‐mer peptides: HVMHKV and HKVAPR. The peptides identified by amino acid substitution of HKVAPR were found to show high affinity and specificity for ZnO nanoparticles. Biotechnol. Bioeng. 2010;106: 845–851. © 2010 Wiley Periodicals, Inc.     

Structural and biological studies on synthetic peptide analogues of a low-affinity calcium-binding site of skeletal troponin C

We have synthesized four oligopeptides that are structural analogues of a low-affinity Ca2+-specific binding site (site II) of rabbit skeletal troponin C. One analogue (peptide 3) was a dodecapeptide with a sequence corresponding to the 12-residue Ca2+-binding loop (residues 63-74 in troponin C), two (peptides 4 and 5) were 23-residue in length, corresponding to residues 52-74 of the protein, and the fourth (peptide 6) was a 25-residue peptide corresponding to residues 50-74. All four peptides had one amino acid substitution within the 12-residue binding loop in which phenylalanine at position 10 was replaced by tyrosine to provide a marker for spectroscopic studies. In addition, peptides 3 and 4 each had a second substitution within the binding loop where glycine at position 6 was replaced by alanine. The second substitution was motivated by the conservation of glycine at the position in the Ca2+-binding loops of all four Ca2+-binding sites in troponin C. The peptides were characterized by their intrinsic fluorescence, ability to enhance the emission of bound Tb3+, affinity for Ca2+ and Tb3+, and circular dichroism. The affinity for Ca2+ was in the range 10-10(2) M-1, and the affinity for Tb3+ was in the range 10(4)-10(5) M-1. The binding constants of the longer peptides were several-fold larger than that of the dodecapeptide. With peptides 4 and 5, substitution of glycine by alanine at position 6 within the 12-residue loop decreased the affinity for Ca2+ by a factor of four, but had little effect on the affinity for Tb3+. However, the mean residue ellipticity of peptide 4 was substantially higher than that of peptide 5. Since peptide 4 differs from peptide 5 only in the substitution of glycine at position 6 in the loop segment, the conservation of glycine at that position may serve a role in providing a suitable secondary structure of the binding sites for interaction with troponin I. Peptides 4 and 6, when present in a large excess, mimic troponin C in regulating fully reconstituted actomyosin ATPase by showing partial calcium sensitivity and activation of the ATPase. Since these peptides are the smallest peptides containing the Ca2+-binding loop of site II, their biological activity suggests that a Ca2+-dependent binding site of troponin C for troponin I could be as short as the segment comprising residues 52-62.     

Molecular Modelling of a Multiphosphorylated Sequence Motif Bound to Hydroxyapatite Surfaces

Proteins and peptides containing the multiphosphorylated motif -Ser(P)-Ser(P)- Ser(P)--Glu-Glu- stabilise amorphous calcium phosphate (ACP) in body fluids and bind with high affinity to crystalline calcium phosphate phases such as hydroxyapatite (HA) regulating crystal growth. Binding of this motif to hydroxyapatite surfaces was investigated in this study using molecular modelling techniques. Using a three-step computational procedure, we have determined the relative binding energies of the motif Ser(P)-Ser(P)-Ser(P)-Glu-Glu to different crystalline surfaces of HA. This analysis revealed preferences of the motif for (100) and (010) surfaces of the crystal and preferences for particular orientations on a given surface. These preferences are principally governed by electrostatic interactions between the crystal lattice and the peptide with the most stable conformers adopting structures where alternate residues exhibit backbone angles characteristic of a -strand and values of an -helix or a distorted -helix, allowing maximal interaction between the acidic side groups and surface calciums. The results of this study are consistent with experimentally-derived data on the interaction of multiphosphorylated proteins/peptides with HA and have implications for the role of these proteins/peptides in calcium phosphate stabilisation and biomineralisation processes.Electronic Supplementary Material available.