Molecular design and optimization of hepatic cancer SLP76‐derived PLCγ1 SH3‐binding peptide with the systematic N‐substitution of peptide PXXP motif

The phospholipase Cγ1 (PLCγ1) is essential for T‐cell signaling and activation in hepatic cancer immune response, which has a regulatory Src homology 3 (SH3) domain that can specifically recognize and interact with the PXXP‐containing decapeptide segment (¹⁸⁵QP P VP P QRPM¹⁹⁴, termed as SLP76^185–194 peptide) of adaptor protein SLP76 following T‐cell receptor ligation. The isolated peptide can only bind to the PLCγ1 SH3 domain with a moderate affinity due to lack of protein context support. Instead of the traditional natural residue mutagenesis that is limited by low structural diversity and shifted target specificity, we herein attempt to improve the peptide affinity by replacing the two key proline residues Pro187 and Pro190 of SLP76^185–194 PXXP motif with nonnatural N‐substituted amino acids, as the proline is the only endogenous N‐substituted amino acid. The replacement would increase peptide flexibility but can restore peptide activity by establishing additional interactions with the domain. Structural analysis reveals that the domain pocket can be divided into a large amphipathic region and a small negatively charged region; they accommodate hydrophobic, aromatic, polar, and moderate‐sized N‐substituted amino acid types. A systematic replacement combination profile between the peptide residues Pro187 and Pro190 is created by structural modeling, dynamics simulation, and energetics analysis, from which six improved and two reduced N‐substituted peptides as well as native SLP76^185–194 peptide are identified and tested for their binding affinity to the recombinant protein of the human PLCγ1 SH3 domain using fluorescence‐based assays. Two N‐substituted peptides, SLP76^185–194(N‐Leu187/N‐Gln190) and SLP76^185–194(N‐Thr187/N‐Gln190), are designed to have high potency (K_d = 0.67 ± 0.18 and 1.7 ± 0.3 μM, respectively), with affinity improvement by, respectively, 8.5‐fold and 3.4‐fold relative to native peptide (K_d = 5.7 ± 1.2 μM).     

Targeting the SH3 domain of human osteoclast‐stimulating factor with rationally designed peptoid inhibitors

Human osteoclast‐stimulating factor (hOSF) is an intracellular protein produced by osteoclasts that induces osteoclast formation and bone resorption. The protein contains a modular Src homology 3 (SH3) domain that mediates the intermolecular recognition and interaction of hOSF with its biological partners. Here, we proposed targeting the hOSF SH3 domain to disrupt hOSF–partner interactions for bone disease therapy by using SH3 inhibitors. In the procedure, the primary sequences of three known hOSF‐interacting proteins (c‐Src, SMN and Sam68) were parsed, from which totally 31 octapeptide segments that contain the core SH3‐binding motif PXXP were extracted, and their binding behavior to hOSF SH3 domain was investigated at structural level using a biomolecular modeling protocol. Several SH3‐binding candidates were identified theoretically and then determined to have high or moderate affinity for the domain using fluorescence spectroscopy assays. One potent peptide ⁴²⁵APP ARP VK⁴³² (K_d = 3.2 μM), which corresponds to the residues 425–432 of Sam68 protein, was used as template to derive N substitution of peptides (peptoids). Considering that proline is the only endogenous N‐substituted amino acid that plays a critical role in SH3–peptide binding, the substitution was addressed at the two key proline residues (Pro427 and Pro430) of the template peptide with nine N‐substituted amino acid types. By systematically evaluating the structural and energetic effects of different N‐substituted amino acids presenting at the two proline sites on peptide binding, we rationally designed five peptoid inhibitors and then determined in vitro their binding affinity to hOSF SH3 domain. Consequently, two designed peptoids APP AR( N ‐Clp) VK and APP AR( N ‐Ffa) VK with Pro430 replaced by N‐Clp and N‐Ffa were confirmed to have increased (K_d = 0.87 μM) and comparable (K_d = 2.9 μM) affinities relative to the template, respectively. In addition, we also found that the Pro427 residue plays an essential role in restricting peptide/peptoid conformations to polyproline II (PPII) helix as the basic requirement of SH3 binding so that the residue cannot be modified. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Crystallographic characterization of the α,γ C12 helix in hybrid peptide sequences

The solid‐state conformations of two αγ hybrid peptides Boc‐[Aib‐γ⁴(R)Ile]₄‐OMe 1 and Boc‐[Aib‐γ⁴(R)Ile]₅‐OMe 2 are described. Peptides 1 and 2 adopt C₁₂‐helical conformations in crystals. The structure of octapeptide 1 is stabilized by six intramolecular 4 → 1 hydrogen bonds, forming 12 atom C₁₂ motifs. The structure of peptide 2 reveals the formation of eight successive C₁₂ hydrogen‐bonded turns. Average backbone dihedral angles for αγ C₁₂ helices are peptide 1 , Aib; φ (°) = ?57.2 ± 0.8, ψ (°) = ?44.5 ± 4.7; γ⁴(R)Ile; φ (°) = ?127.3 ± 7.3, θ₁ (°) = 58.5 ± 12.1, θ₂ (°) = 67.6 ± 10.1, ψ (°) = ?126.2 ± 16.1; peptide 2 , Aib; φ (°) = ?58.8 ± 5.1, ψ (°) = ?40.3 ± 5.5; ψ⁴(R)Ile; φ (°) = ?123.9 ± 2.7, θ₁ (°) = 53.3 θ 4.9, θ ₂ (°) = 61.2 ± 1.6, ψ (°) = ?121.8 ± 5.1. The tendency of γ⁴‐substituted residues to adopt gauche–gauche conformations about the C^α–C^β and C^β–C^γ bonds facilitates helical folding. The αγ C₁₂ helix is a backbone expanded analog of α peptide 3₁₀ helix. The hydrogen bond parameters for α peptide 3₁₀ and α‐helices are compared with those for αγ hybrid C₁₂ helix. Copyright © 2016 European Peptide Society and John Wiley & Sons.     

Structure‐antigenicity of the V3 region of SIVmac envelope glycoprotein

The objective of this study was to analyze the immunogenicity and antigenicity of the V3 domain (Cys313–Cys346) of the external envelope glycoprotein gp125 of SIVmac251. The corresponding peptide was synthesized and characterized as linear and cyclic peptides. Our results showed that this region, as for HIV‐1, contained an immunodominant epitope. The antigenicity was similar for the linear and cyclic peptides when tested against a panel of 15 sera from SIV infected macaques. Similarly, both peptide structures presented similar immunogenicity as shown by the characterization of the anti‐peptide antibodies produced in rabbits against the cyclic and linear forms. But, unexpectedly, the antibodies produced against linear peptides recognized with a relatively higher intensity the native envelope gp140 than those produced against the cyclic structure. Furthermore, we showed that these antibodies recognized better the deglycosylated form of the glycoprotein. But, in contrast to the neutralizing activity obtained with anti‐V3 peptides from HIV‐1, no antiviral activity was obtained with antibodies generated against linear or cyclic SIVmac V3 peptides. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Syntheses and activities of backbone‐side chain cyclic octapeptide ligands with N‐functionalized phosphotyrosine for the N‐terminal SH2‐domain of the protein tyrosine phosphatase SHP‐1

The protein tyrosine phosphatase SHP‐1 plays an important role in many physiological and pathophysiological processes. This phosphatase is activated through binding of ligands to its SH2‐domains, mainly to the N‐terminal one. Based on a theoretical docking model, backbone‐to‐side chain cyclized octapeptides were designed as ligands. Assembly of such modelled structures required the synthesis of N‐functionalized tyrosine derivatives and their incorporation into the sequence. Because of difficulties encountered in the condensation of N‐protected amino acids to the N‐alkylated tyrosine‐peptide we synthesized and used preformed dipeptide building units. As all attempts to obtain phosphorylated dipeptide units failed, the syntheses had to be performed with a free phenolic function. Use of different N‐alkyl or cycloalkyl residues in the N‐functionalized side chains allowed to investigate the effect of ring size, flexibility and hydrophobicity of formed lactam bridges on stimulatory activity. All tested linear and cyclic octapeptides stimulate the phosphatase activity of SHP‐1. Stimulatory activities of cyclic ligands increase with the chain length of the lactam bridges resulting in increased flexibility and better entropic preformation of the binding conformation. The strong activity of some cyclic octapeptides supports the modelled structure. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Exploring the systematic effect of N-substituted PxxP motifs on peptoid affinity to ARHGEF5/TIM SH3 domain and its relationship with ARHGEF5/TIM activation

The TIM protein is a short isoform of full-length Rho guanine nucleotide exchange factor 5 (ARHGEF5), which acts as a functional regulator of Rho-dependent signaling pathways by activating the Rho family of GTPases. The activation is auto-inhibited by a putative helix N-terminal to the DH domain of TIM, which is stabilized by the intramolecular interaction of C-terminal SH3 domain with a proline-rich region ⁴⁷SSPRQP RKAL⁵⁶ (termed as SSP peptide) between the putative helix and the DH domain. Previously, we demonstrate that the auto-inhibitory state of TIM protein can be relieved by targeting its SH3 domain with rationally designed peptide ligands. However, the designed natural peptides have only a moderately increased affinity (~2-fold) as compared to the cognate SH3-SSP interaction and are susceptible to protease degradation. Here, considering that proline is the only endogenous N-substituted amino acid that plays a critical role in SH3-peptide recognition, the two key proline residues Pro49 and Pro52 in the core ⁴⁹PxxP ⁵² motif of SSP peptide are systematically replaced by 19 N-substituted amino acid types to derive a variety of nonnatural peptoid ligands for TIM SH3 domain. Dynamics and energetics analyses reveal that the replacement would impair the active polyproline II (PPII) helical conformation of SSP peptide due to lack of structural constraint introduced by the five-membered ring of native proline side-chains, thus increasing the peptide flexibility that could incur a large entropy penalty upon binding to the domain. However, the impairment is not very significant and the peptide affinity may also be restored and improved if the N-substituted motif of derived peptiod ligands can effectively interact with the PxxP-binding site of TIM SH3 domain. Consequently, a number of potent peptoids are successfully designed by fluorescence spectroscopy confirmation, in which three (ie, SSP[N-Ile49, N-Asn52], SSP[N-Phe49, N-Gln52], and SSP[N-Tyr49, N-Asn52]) exhibit considerably increased affinity (K_d = 0.09, 0.07, and 0.04 μM, respectively) relative to the native SSP peptide (K_d = 0.87 μM). In addition, guanine nucleotide exchange assays also substantiate that the designed SH3-targeted peptiods can effectively enhance TIM-catalyzed RhoA exchange activity (EA), which is observed to present an exponential relationship with the measured SH3-peptoid binding affinity (pK_d).     

Synthesis and evaluation of conformationally constrained peptide analogues as the Src SH3 domain binding ligands

Src kinase activity is regulated by the interaction of SH3 domain with protein sequences that are rich in proline residues. Identification of more potent SH3 domain binding ligands that can regulate Src kinase activity is a subject of major interest. Conformationally constrained peptides have been previously used for improving the binding potency of the Src SH2 domain binding peptide ligands and peptide substrates of the substrate-binding site of Src. A series of peptide analogues of Ac-VSLARRPLPPLP (1, Ac-VSL12, K_d = 0.34 μM) were synthesized by introducing conformational constraints to improve the binding affinity towards the Src SH3 domain. Peptides synthesized through cyclization between N-terminal to C-terminal [VSLARRPLPPLP] or N-terminal to side chain flanking residues (i.e., [^βAVS]LARRPLPPLP and [VSLE]RRPLPPLP) exhibited at least 6.4-fold less binding affinity (K_d = 2.19–4.85 μM) when compared to 1. The data suggest upon N-terminal cyclization with C-terminal or flanking residues, the interactions of the amino acids in the core RPLPPLP reduce significantly with the residues within the Src SH3 domain. Conformationally constrained peptide V[SLARRPLPPLP] (5) was synthesized through cyclization of C-terminal to the serine side chain and displayed a comparable binding affinity (K_d = 0.35 μM) towards the Src SH3 domain versus that of 1. Thus, this template may be used to optimize and generate more potent analogues with higher stability.     

Examination of the active secondary structure of the peptide 101.10, an allosteric modulator of the interleukin‐1 receptor,by positional scanning using β‐amino γ‐lactams

The relationship between the conformation and biological activity of the peptide allosteric modulator of the interleukin‐1 receptor 101.10 (D ‐Arg‐D ‐Tyr‐D ‐Thr‐D ‐Val‐D ‐Glu‐D ‐Leu‐D ‐Ala‐NH₂) has been studied using (R)‐ and (S)‐Bgl residues. Twelve Bgl peptides were synthesized using (R)‐ and (S)‐cyclic sulfamidate reagents derived from L ‐ and D ‐aspartic acid in an optimized Fmoc‐compatible protocol for efficient lactam installment onto the supported peptide resin. Examination of these (R)‐ and (S)‐Bgl 101.10 analogs for their potential to inhibit IL‐1β‐induced thymocyte cell proliferation using a novel fluorescence assay revealed that certain analogs exhibited retained and improved potency relative to the parent peptide 101.10. In light of previous reports that Bgl residues may stabilize type II′β‐turn‐like conformations in peptides, CD spectroscopy was performed on selected compounds to identify secondary structure necessary for peptide biological activity. Results indicate that the presence of a fold about the central residues of the parent peptide may be important for activity. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Modulation of SHP‐1 phosphatase activity by monovalent and bivalent SH2 phosphopeptide ligands

A sequence derived from the epithelial receptor tyrosine kinase Ros (pY2267) represents a high‐affinity binding partner for protein tyrosine phosphatase SHP‐1 and was recently used as lead structure to analyze the recognition requirements for the enzyme's N‐SH2 domain. Here, we focused on a set of peptides comprising C‐terminally extended linear and conformationally constrained side chain‐bridged cyclic N‐SH2 ligands based on the consensus sequence LxpYhxh(h/b)(h/b) (x = any amino acid, h = hydrophobic, and b = basic residue). Furthermore, the bivalent peptides described were designed to modulate the activity of SHP‐1 through binding to both, the N‐SH2 domain as well as an independent binding site on the surface of the catalytic domain (PTP domain). Consistent with previous experimental findings, surface plasmon resonance experiments revealed dissociation constants of most compounds in the low micromolar range. One peptide, EGLNpYc[KVD]MFPAPEEE? NH₂, displayed favorable binding affinity, but reduced ability to stimulate SHP‐1. Docking experiments revealed that the binding of this ligand occurs in binding mode I, recently described to lead to an inhibited activation of SHP‐1. In summary, results presented in this study suggest that inhibitory N‐SH2 ligands of SHP‐1 may be obtained by designing bivalent compounds that associate with the N‐SH2 domain and simultaneously occupy a specific binding site on the PTP domain. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 102–112, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Basic conformers in β‐peptides

The conformation of oligomers of β‐amino acids of the general type Ac‐[β‐Xaa]_n‐NHMe (β‐Xaa = β‐Ala, β‐Aib, and β‐Abu; n = 1–4) was systematically examined at different levels of ab initio molecular orbital theory (HF/6‐31G*, HF/3‐21G). The solvent influence was considered employing two quantum‐mechanical self‐consistent reaction field models. The results show a wide variety of possibilities for the formation of characteristic elements of secondary structure in β‐peptides. Most of them can be derived from the monomer units of blocked β‐peptides with n = 1. The stability and geometries of the β‐peptide structures are considerably influenced by the side‐chain positions, by the configurations at the C^α‐ and C^β‐atoms of the β‐amino acid constituents, and especially by environmental effects. Structure peculiarities of β‐peptides, in particular those of various helix alternatives, are discussed in relation to typical elements of secondary structure in α‐peptides. © 1999 John Wiley & Sons, Inc. Biopoly 50: 167–184, 1999     

Epitope analysis of the multiphosphorylated peptide αs1‐casein(59–79)

The multiphosphorylated tryptic peptide α_s1‐casein(59–79) has been shown to be antigenic with anti‐casein antibodies. In an approach to determine the amino acyl residues critical for antibody binding we undertook an epitope analysis of the peptide using overlapping synthetic peptides. With α_s1‐casein(59–79) as the adsorbed antigen in a competitive ELISA only two of five overlapping synthetic peptides at 1 mM significantly inhibited binding of the anti‐casein antibodies. Peptides Glu‐Ser(P)‐Ile‐Ser(P)‐Ser(P)‐Ser(P)‐Glu‐Glu and Ile‐Val‐Pro‐Asn‐Ser(P)‐Val‐Glu‐Glu inhibited antibody binding by 20.0±3.6% and 60.3±7.9%, respectively. The epitope of Glu⁶³‐Ser(P)‐Ile‐Ser(P)‐Ser(P)‐Ser(P)‐Glu‐Glu⁷⁰ was further localised to the phosphoseryl cluster as the peptide Ser(P)‐Ser(P)‐Ser(P) significantly inhibited binding of the anti‐casein antibodies to α_s1‐casein(59–79) by 29.5±7.4%. Substitution of Ser(P)⁷⁵ with Ser⁷⁵ in the second inhibitory peptide Ile‐Val‐Pro‐Asn‐Ser(P)⁷⁵‐Val‐Glu‐Glu also abolished inhibition of antibody binding to α_s1‐casein (59–79) demonstrating that Ser(P)⁷⁵ is also a critical residue for recognition by the antibodies. These data show that the phosphorylated residues in the cluster sequence ‐Ser(P)⁶⁶‐Ser(P)‐Ser(P)⁶⁸ and in the sequence ‐Pro⁷³‐Asn‐Ser(P)‐Val‐Glu⁷⁷‐ are critical for antibody binding to α_s1‐casein(59–79) and further demonstrate that a highly phosphorylated segment of a protein can be antigenic. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Stability and folding of the SH3 domain of bruton's tyrosine kinase

Bruton's tyrosine kinase (BTK) plays an important role in B cell development. Deletion of C-terminal 14 amino acids of the SH3 domain of BTK results in X-linked agammaglobulinemia (XLA), an inherited disease. We report here on the stability and folding of SH3 domain of BTK. Peptides corresponding to residues 216–273 (58 residues) and 216–259 (44 residues) of BTK SH3 domain were synthesized by solid phase methods; the first peptide constitutes the entire SH3 domain of BTK while the latter peptide lacks 14 amino acid residues of the C-terminal. The 58 amino acid peptide forms mainly a β-barrel type folding unit. Although small and lacking disulfide bonds, this peptide is extremely stable to thermal denaturation. Based on circular dichroism measurements, its melting temperature was found to be high, 82°C at pH 6.0. However, the Gibbs free energy (ΔG) of the intrinsic stability and thermodynamic spontaneity of unfolding were found to be low, 2.6 kcal/mol by Gdn·HCl denaturation experiments, as compared to 12 kcal/mol obtained for larger single domain proteins, indicating poor stability of SH3 domain. Addition of 500 mM of Na₂SO₄ increased the free energy change ΔG to 4.0 kcal/mol, suggesting an ionic strength effect. The truncated peptide fails to fold correctly and adopts random coil conformation in contrast to 58 amino acid β-barrel peptide, which exhibits high thermal stability but normal or low stability at ambient temperature. These results, to our knowledge the first to delineate the importance of C-terminal in structural integrity of SH3 domains, indicate also that improper folding and/or poor stability of mutant SH3 domain in BTK likely causes XLA. Proteins 28:465–471 © 1996 Wiley-Liss, Inc.     

β‐Amino acids containing peptides and click‐cyclized peptide as β‐turn mimics: a comparative study with ‘conventional’ lactam‐ and disulfide‐bridged hexapeptides

The increasing interest in click chemistry and its use to stabilize turn structures led us to compare the propensity for β‐turn stabilization of different analogs designed as mimics of the β‐turn structure found in tendamistat. The β‐turn conformation of linear β‐amino acid‐containing peptides and triazole‐cyclized analogs were compared to ‘conventional’ lactam‐ and disulfide‐bridged hexapeptide analogs. Their 3D structures and their propensity to fold in β‐turns in solution, and for those not structured in solution in the presence of α‐amylase, were analyzed by NMR spectroscopy and by restrained molecular dynamics with energy minimization. The linear tetrapeptide Ac‐Ser‐Trp‐Arg‐Tyr‐NH₂ and both the amide bond‐cyclized, c[Pro‐Ser‐Trp‐Arg‐Tyr‐D ‐Ala] and the disulfide‐bridged, Ac‐c[Cys‐Ser‐Trp‐Arg‐Tyr‐Cys]‐NH₂ hexapeptides adopt dominantly in solution a β‐turn conformation closely related to the one observed in tendamistat. On the contrary, the β‐amino acid‐containing peptides such as Ac‐(R)‐β³‐hSer‐(S)‐Trp‐(S)‐β³‐hArg‐(S)‐β³‐hTyr‐NH₂, and the triazole cyclic peptide, c[Lys‐Ser‐Trp‐Arg‐Tyr‐βtA]‐NH₂, both specifically designed to mimic this β‐turn, do not adopt stable structures in solution and do not show any characteristics of β‐turn conformation. However, these unstructured peptides specifically interact in the active site of α‐amylase, as shown by TrNOESY and saturation transfer difference NMR experiments performed in the presence of the enzyme, and are displaced by acarbose, a specific α‐amylase inhibitor. Thus, in contrast to amide‐cyclized or disulfide‐bridged hexapeptides, β‐amino acid‐containing peptides and click‐cyclized peptides may not be regarded as β‐turn stabilizers, but can be considered as potential β‐turn inducers. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Docking and molecular dynamics simulations of the Fyn‐SH3 domain with free and phospholipid bilayer‐associated 18.5‐kDa myelin basic protein (MBP)—Insights into a noncanonical and fuzzy interaction

The molecular details of the association between the human Fyn‐SH3 domain, and the fragment of 18.5‐kDa myelin basic protein (MBP) spanning residues S38–S107 (denoted as xα2‐peptide, murine sequence numbering), were studied in silico via docking and molecular dynamics over 50‐ns trajectories. The results show that interaction between the two proteins is energetically favorable and heavily dependent on the MBP proline‐rich region (P93‐P98) in both aqueous and membrane environments. In aqueous conditions, the xα2‐peptide/Fyn‐SH3 complex adopts a “sandwich”"‐like structure. In the membrane context, the xα2‐peptide interacts with the Fyn‐SH3 domain via the proline‐rich region and the β‐sheets of Fyn‐SH3, with the latter wrapping around the proline‐rich region in a form of a clip. Moreover, the simulations corroborate prior experimental evidence of the importance of upstream segments beyond the canonical SH3‐ligand. This study thus provides a more‐detailed glimpse into the context‐dependent interaction dynamics and importance of the β‐sheets in Fyn‐SH3 and proline‐rich region of MBP. Proteins 2017; 85:1336–1350. © 2017 Wiley Periodicals, Inc.     

Probing the interaction between cHAVc3 peptide and the EC1 domain of E-cadherin using NMR and molecular dynamics simulations

The goal of this work is to probe the interaction between cyclic cHAVc3 peptide and the EC1 domain of human E-cadherin protein. Cyclic cHAVc3 peptide (cyclo(1,6)Ac-CSHAVC-NH₂) binds to the EC1 domain as shown by chemical shift perturbations in the 2D ¹H,-¹⁵N-HSQC NMR spectrum. The molecular dynamics (MD) simulations of the EC1 domain showed folding of the C-terminal tail region into the main head region of the EC1 domain. For cHAVc3 peptide, replica exchange molecular dynamics (REMD) simulations generated five structural clusters of cHAVc3 peptide. Representative structures of cHAVc3 and the EC1 structure from MD simulations were used in molecular docking experiments with NMR constraints to determine the binding site of the peptide on EC1. The results suggest that cHAVc3 binds to EC1 around residues Y36, S37, I38, I53, F77, S78, H79, and I94. The dissociation constants (K_d values) of cHAVc3 peptide to EC1 were estimated using the NMR chemical shifts data and the estimated K_ds are in the range of .5 × 10^?5–7.0 × 10^?5 M.     

Design of Peptides Using α,β-Dehydro-residues: Synthesis,Crystal Structure and Molecular Conformation of N-Boc-L-Val-ΔPhe–ΔPhe-L-Ala-OCH3

To obtain general rules of peptide design using α,β-dehydro-residues, a sequence with two consecutive ΔPhe-residues, Boc-L -Val-ΔPhe–ΔPhe- L -Ala-OCH₃, was synthesized by azlactone method in solution phase. The peptide was crystallized from its solution in an acetone/water mixture (70:30) in space group P6₁ with a=b=14.912(3) Å, c= 25.548(5) Å, V=4912.0(6) ų. The structure was determined by direct methods and refined by a full matrix least-squares procedure to an R value of 0.079 for 2891 observed [I?3σ(I)] reflections. The backbone torsion angles ?₁=?54(1)°, ψ₁= 129(1)°, ω₁=?177(1)°, ?₂ =57(1)°, ψ₂=15(1)°, ω₂ =?170(1)°, ?₃=80(1)°, ψ₃ =7(2)°, ω₃=?177(1)°, ?₄ =?108(1)° and ψ^T₄=?34 (1)° suggest that the peptide adopts a folded conformation with two overlapping β-turns of types II and III′. These turns are stabilized by two intramolecular hydrogen bonds between the CO of the Boc group and the NH of ΔPhe³ and the CO of Val¹ and the NH of Ala⁴. The torsion angles of ΔPhe² and ΔPhe³ side chains are similar and indicate that the two ΔPhe residues are essentially planar. The folded molecules form head-to- tail intermolecular hydrogen bonds giving rise to continuous helical columns which run parallel to the c-axis. This structure established the formation of two β-turns of types II and III′ respectively for sequences containing two consecutive ΔPhe residues at (i+2) and (i+3) positions with a branched β-carbon residue at one end of the tetrapeptide.     

Epimerization‐free synthesis of cyclic peptide by use of the O‐acyl isopeptide method

A head‐to‐tail cyclization of a protected linear hexapeptide with a C‐terminal O‐acyl isopeptide proceeded to give a cyclic O‐acyl isopeptide without epimerization. The cyclic O‐acyl isopeptide possessed different secondary structures compared with the native cyclic peptide. The isopeptide was then efficiently converted to the desired cyclic peptide via an O‐to‐N acyl migration reaction using a silica gel‐anchored base. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Site‐specific inhibition of integrin αvβ3‐vitronectin association by a ser‐asp‐val sequence through an Arg‐Gly‐Asp‐binding site of the integrin

A functional proteomic technology using protein chip and molecular simulation was used to demonstrate a novel biomolecular interaction between P11, a peptide containing the Ser‐Asp‐Val (SDV) sequence and integrin αvβ3. P11 (HSDVHK) is a novel antagonistic peptide of integrin αvβ3 screened from hexapeptide library through protein chip system. An in silico docking study and competitive protein chip assay revealed that the SDV sequence of P11 is able to create a stable inhibitory complex onto the vitronectin‐binding site of integrin αvβ3. The Arg‐Gly‐Asp (RGD)‐binding site recognition by P11 was site specific because the P11 was inactive for the complex formation of a denatured form of integrin–vitronectin. P11 showed a strong antagonism against αvβ3‐GRGDSP interaction with an IC₅₀ value of 25.72±3.34 nM, whereas the value of GRGDSP peptide was 1968.73±444.32 nM. The binding‐free energies calculated from the docking simulations for each P11 and RGD peptide were ?3.99 and ?3.10 kcal/mol, respectively. The free energy difference between P11 and RGD corresponds to approximately a 4.5‐fold lower K_i value for the P11 than the RGD peptide. The binding orientation of the docked P11 was similar to the crystal structure of the RGD in αvβ3. The analyzed docked poses suggest that a divalent metal–ion coordination was a common driving force for the formation of both SDV/αvβ3 and RGD/αvβ3 complexes. This is the first report on the specific recognition of the RGD‐binding site of αvβ3 by a non‐RGD containing peptide using a computer‐assisted proteomic approach.     

Isostructural unbranched alkyl‐chains as tools for stabilizing β‐turn structure

To investigate the structural role played by isostructural unbranched alkyl‐chains on the conformational ensemble and stability of β‐turn structures, the conformational properties of a designed model peptide: Plm‐Pro‐Gly‐Pda ( 1 , Plm: H₃C—(CH₂)₁₄—CONH—; Pda: —CONH— (CH₂)₁₄—CH₃) have been examined and compared with the parent peptide: Boc‐Pro‐Gly‐NHMe ( 2 , Boc: tert‐butoxycarbonyl; NHMe: N‐methylamide). The characteristic ¹³C NMR chemical‐shifts of the Pro C^β and C^γ resonances ascertained the incidence of an all‐trans peptide‐bond in low polarity deuterochloroform solution. Using FTIR and ¹H NMR spectroscopy, we establish that apolar alkyl‐chains flanking a β‐turn promoting Pro‐Gly sequence impart definite incremental stability to the well‐defined hydrogen‐bonded structure. The assessment of ¹H NMR derived thermodynamic parameters of the hydrogen‐bonded amide‐NHs via variable temperature indicate that much weaker hydrophobic interactions do contribute to the stability of folded reverse turn structures. The far‐UV CD spectral patterns of 1 and 2 in 2,2,2‐trifluoroethanol are consistent with Pro‐Gly specific type II β‐turn structure, concomitantly substantiate that the flanking alkyl‐chains induce substantial bias in enhanced β‐turn populations. In view of structural as well as functional importance of the Pro‐Gly mediated secondary structures, besides biochemical and biological significance of proteins lipidation via myristoylation or palmytoilation, we highlight potential convenience of the unbranched Plm and Pda moieities not only as main‐chain N‐ and C‐terminal protecting groups but also to mimic and stabilize specific isolated secondary and supersecondary structural components frequently observed in proteins and polypeptides. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 419–426, 2013.     

Nanostructures from the self‐assembly of α‐helical peptide amphiphiles

Self‐assembly of PAs composed of palmitic acid and several repeated heptad peptide sequences, C₁₅H₃₁CO‐(IEEYTKK)_n‐NH₂ (n = 1–4, represented by PA1–PA4), was investigated systematically. The secondary structures of the PAs were characterized by CD. PA3 and PA4 (n = 3 and 4, respectively) showed an α‐helical structure, whereas PA1 and PA2 (n = 1 and 2, respectively) did not display an α‐helical conformations under the tested conditions. The morphology of the self‐assembled peptides in aqueous medium was studied by transmission electron microscopy. As the number of heptad repeats in the PAs increased, the nanostructure of the self‐assembled peptides changed from nanofibers to nanovesicles. Changes of the secondary structures and the self‐assembly morphologies of PA3 and PA4 in aqueous medium with various cations were also studied. The critical micelle concentrations were determined using a pyrene fluorescence probe. In conclusion, this method may be used to design new peptide nanomaterials. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.