Artificial peptides with unnatural components designed for materializing protein function

In order to design functional peptides, we employed two strategies. The first one is to incorporate rigid unnatural amino acids into peptides to make the peptide backbone rigid. Functions were expected to appear through the conformational control by the strategy. A series of cyclic peptides constituted of alternating natural amino acids and 3-aminobenzoic acid, used as an unnatural amino acid, were synthesized. These cyclic peptides were found to function as strong binders for phosphomonoester, catalysts for ester hydrolysis, and/or ion channels. The second strategy is to conjugate peptides with unnatural and inherently functional molecules. Following this strategy, oligo(L-leucine)- or oligo(L-phenylalanine)-modified ruthenium tris(bipyridine) complexes were synthesized. Distance dependence of the photoinduced electron transfer from the ruthenium complexes and the function as sensors for phosphate anion (H(2)PO(-)(4)) are discussed.     

Protease-activated receptor 4-like peptides bind to thrombin through an optimized interaction with the enzyme active site surface

Protease-activated receptor 4 (PAR4) is cleaved by thrombin at the R47-G48 peptide bond. Unlike PAR1, PAR4 does not contain a sequence readily predicted to interact with thrombin anion binding exosite-I. HPLC kinetic results on hydrolysis of PAR4 peptides (38-51 and 38-62) reveal that extending the sequence from the active site toward the exosite does not promote further binding interactions with thrombin. One-dimensional-proton line-broadening NMR indicates that the amino acids occupying the P(4)-P(1) positions of PAR4 (38-47), 44PAPR(47), come into direct contact with the thrombin surface. Less contact arises from the Leu43 at the P(5) position. Two-dimensional total correlation spectroscopy and two-dimensional transferred nuclear Overhauser effect spectroscropy studies on this complex reveal that Leu43 is flexible and can exhibit two conformational states. The binding mode observed for PAR4 peptides is similar to that of PAR1 peptides. PAR4 takes advantage of a distinctive sequence to optimize its interactions with the thrombin active site surface.     

The Carboxy Terminus of the Ligand Peptide Determines the Stability of the MHC Class I Molecule H-2Kb: A Combined Molecular Dynamics and Experimental Study

Major histocompatibility complex (MHC) class I molecules (proteins) bind peptides of eight to ten amino acids to present them at the cell surface to cytotoxic T cells. The class I binding groove binds the peptide via hydrogen bonds with the peptide termini and via diverse interactions with the anchor residue side chains of the peptide. To elucidate which of these interactions is most important for the thermodynamic and kinetic stability of the peptide-bound state, we have combined molecular dynamics simulations and experimental approaches in an investigation of the conformational dynamics and binding parameters of a murine class I molecule (H-2K^b) with optimal and truncated natural peptide epitopes. We show that the F pocket region dominates the conformational and thermodynamic properties of the binding groove, and that therefore the binding of the C terminus of the peptide to the F pocket region plays a crucial role in bringing about the peptide-bound state of MHC class I.     

Exploration of structural features of monomeric helical peptides designed with a genetic algorithm

A genetic algorithm (GA)-based strategy to dissect the determinants of peptide folding into alpha-helix was developed. The structural information of helical peptides was obtained with respect to patterns of sequence variability. In many previously reported studies the intrinsic alpha-helical propensities of amino acids although sequence-dependent are apparently independent of the amino acid position. In this research, monomeric helical peptides selected from possible sequences produced by a GA-chemical synthesis were analyzed to identify possible influential structural features. These hexadeca-peptides were obtained after four successive generations. A total of 128 synthetic peptides were evaluated via circular dichroism (CD) measurements in aqueous solution, while the mean ellipticity at 222 nm confirmed the monomeric state of the peptides. The results presented here show that our GA-based strategy may be useful in the design of proteins with increased alpha-helix content.     

Model peptide‐based system used for the investigation of metal ions binding to histidine‐containing polypeptides

The reaction of histidine‐containing polypeptides with toxic and essential metals and the molecular mechanism of complexation has yet to be determined, particularly with respect to the conformational changes of the interacting macromolecules. Therefore, a system of oligopeptides containing histidine residues in various positions of Ala or Gly sequences has been designed and used in heavy metal comparatively binding experiments. The role of spacing residues (Gly and Ala repeats) in selecting the various conformations was investigated. The newly synthesized peptides and metal ion adducts have been characterized by Fourier transform infrared spectroscopy (FTIR) as well as electrospray ion trap mass spectrometry (ESI–MS) and circular dichroism (CD). The analysis of CD‐spectra of the four peptides in water revealed that the secondary structure depends much on the position of each amino acid in the peptide backbone. Our peptides system reveals various binding mechanisms of metal ions to peptides depending on the position of histidine residue and the corresponding conformations of Ala or Gly sequences. Biological and medical consequences of conformational changes of metal‐bound peptides are further discussed. Thus, the binding of heavy metals to four peptides may serve as a model system with respect to the conformational consequences of the metal addition on the amino acid repeats situated in prion protein. © 2010 Wiley Periodicals, Inc. Biopolymers 93:497–508, 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     

Synthetic peptides from the N-domains of CEACAMs activate neutrophils.

Four members of the carcinoembryonic antigen family, CEACAM1, CEACAM8, CEACAM6 and CEACAM3, recognized by CD66a, CD66b, CD66c and CD66d monoclonal antibodies (mAb), respectively, are expressed on human neutrophils. CD66a, CD66b, CD66c and CD66d mAb binding to neutrophils triggers an activation signal that regulates the adhesive activity of CD11/CD18, resulting in an increase in neutrophil adhesion to human umbilical vein endothelial cells. Molecular modeling of CEACAM1 using IgG and CD4 as models has been performed, and three peptides from the N-terminal domain were found to increase neutrophil adhesion to human umbilical vein endothelial cell monolayers. The peptides were 14 amino acids in length and were predicted to be present at loops and turns between beta-sheets. To better understand the amino acid sequences critical for this biological activity, in the present study we examined the other neutrophil CEACAMs and the highly homologous CEACAM, CEA. Molecular modeling of the N-terminal domains of human CEACAM8, -6, -3 and CEA was performed. Twenty peptides, each 14 amino acids in length, that were homologous to the previously reported peptides from the N-domains of CEACAM1, were synthesized and tested for their ability to alter neutrophil adhesion. Only one new peptide, from the N-domain of CEA, was found to increase neutrophil adhesion, and this peptide differed from the corresponding CEACAM1 peptide by only a single conservative amino acid substitution. Importantly, minor amino acid differences between active and inactive homologous peptides suggest regions of these peptides that are critical for biological activity. The data suggest that the regions SMPF of peptide CD66a-1, QLFG of peptide CD66a-2 and NRQIV of peptide CD66a-3 are critical for the activities of these peptides, and for the native CEACAMs.     

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.     

In vivo phosphorylation of a recombinant peptide substrate of CDPK suggests involvement of CDPK in plant stress responses

A phage display library displaying random peptides 15 amino acids in length was screened for peptides that interact with soybean (Glycine max L.) CDPKalpha, an isoform of calcium-dependent protein kinase (EC 2.7.1.37). Interaction of phage displaying the peptide RHPTLTRSPTLRNIQ with CDPKalpha was confirmed in an independent binding assay. A synthetic peptide corresponding to this sequence plus the surrounding amino acids AERHPTLTRSPTLRNIQPPC was synthesized and found to be a substrate of CDPK isoforms alpha, beta, and gamma. A second random peptide phage display library was constructed that displayed the substrate peptide sequence plus an additional 10 random amino acids on its amino-terminal side. Nine new peptides were obtained from the screening, all of which were phosphorylated by CDPKalpha. Sequence VSPRSFWTTWRHPTLTRSPTLRNIQ appeared twice in the screen. Because it agreed well with the consensus phosphorylation site of CDPKs, its coding sequence was cloned and stably transformed into tobacco cells. The substrate peptide expressed in tobacco was phosphorylated by recombinant CDPKalpha in vitro and by endogenous CDPK in vivo. Increased phosphorylation of the peptide substrate in response to hydrogen peroxide treatment was observed in transgenic tobacco cells. These results show that the peptide substrate expressed in tobacco cells can be used as a CDPK activity reporter for in vivo studies.     

Conformational studies of a short linear peptide corresponding to a major conserved neutralizing epitope of human respiratory syncytial virus fusion glycoprotein

The conformational properties of a 21-residue peptide, corresponding to amino acids 255 to 275 (F255-275) of the human respiratory syncytial virus fusion (F) glycoprotein, have been studied by CD and nmr spectroscopy. This peptide includes residues 262, 268, and 272 of the F polypeptide that are essential for integrity of most epitopes that mapped into a major antigenic site of the F molecule. CD data indicate that F255-275 adopts a random coil conformation in aqueous solution at low peptide concentrations. However, as the concentration of peptide is increased, a higher percentage of peptide molecules adopts an organized structure. This effect can be more easily observed when trifluoroethanol (30%) is added to peptide solutions, giving rise to CD spectra that resemble those of α-helix structures. These conformational changes were confirmed by nmr spectroscopy. The nuclear Overhauser effects observed in 30% trifluoroethanol/water together with the conformational H_α chemical shift data allowed us to propose a structural model of helix-loop-helix for the peptide in solution. In addition, these helical regions contain the amino acid residues essential for epitope integrity in the native F molecule. These results give new insights into the antigenic structure of the respiratory syncytial virus F glycoprotein. © 1996 John Wiley & Sons, Inc.     

Conformational Flexibility Determines Selectivity and Antibacterial, Antiplasmodial, and Anticancer Potency of Cationic ��-Helical Peptides

We used a combination of fluorescence, circular dichroism (CD), and NMR spectroscopies in conjunction with size exclusion chromatography to help rationalize the relative antibacterial, antiplasmodial, and cytotoxic activities of a series of proline-free and proline-containing model antimicrobial peptides (AMPs) in terms of their structural properties. When compared with proline-free analogs, proline-containing peptides had greater activity against Gram-negative bacteria, two mammalian cancer cell lines, and intraerythrocytic Plasmodium falciparum, which they were capable of killing without causing hemolysis. In contrast, incorporation of proline did not have a consistent effect on peptide activity against Mycobacterium tuberculosis. In membrane-mimicking environments, structures with high α-helix content were adopted by both proline-free and proline-containing peptides. In solution, AMPs generally adopted disordered structures unless their sequences comprised more hydrophobic amino acids or until coordinating phosphate ions were added. Proline-containing peptides resisted ordering induced by either method. The roles of the angle subtended by positively charged amino acids and the positioning of the proline residues were also investigated. Careful positioning of proline residues in AMP sequences is required to enable the peptide to resist ordering and maintain optimal antibacterial activity, whereas varying the angle subtended by positively charged amino acids can attenuate hemolytic potential albeit with a modest reduction in potency. Maintaining conformational flexibility improves AMP potency and selectivity toward bacterial, plasmodial, and cancerous cells while enabling the targeting of intracellular pathogens.     

"De novo" design of peptides with specific lipid-binding properties

In this study, we describe an in silico method to design peptides that can be made of non-natural amino acids and elicit specific membrane-interacting properties. The originality of the method holds in the capacities developed to design peptides from any non-natural amino acids as easily as from natural ones, and to test the structure stability by an angular dynamics rather than the currently-used molecular dynamics. The goal of this study was to design a non-natural tilted peptide. Tilted peptides are short protein fragments able to destabilize lipid membranes and characterized by an asymmetric distribution of hydrophobic residues along their helix structure axis. The method is based on the random generation of peptides and their selection on three main criteria: mean hydrophobicity and the presence of at least one polar residue; tilted insertion at the level of the acyl chains of lipids of a membrane; and conformational stability in that hydrophobic phase. From 10,000,000 randomly-generated peptides, four met all the criteria. One was synthesized and tested for its lipid-destabilizing properties. Biophysical assays showed that the "de novo" peptide made of non-natural amino acids is helical either in solution or into lipids as tested by Fourier transform infrared spectroscopy and is able to induce liposome fusion. These results are in agreement with the calculations and validate the theoretical approach.     

CD4-derived synthetic peptide blocks the binding of HIV-1 GP120 to CD4-bearing cells and prevents HIV-1 infection

The T cell surface glycoprotein CD4 plays an important role in mediating cellular immunity and serves as the receptor for human immunodeficiency virus. In order to identify primary sequences within the CD4 molecule that may be involved in the binding of the HIV-I envelope, we synthesized various peptides corresponding to the V1, V2, V3, and V4 domains of CD4. We tested the ability of these peptides to block the binding of purified HIV-I gp120 to CD4+ human lymphoblastic leukemia cells (CEM) using fluorescence-activated cell sorting. One of these peptides, corresponding to CD4 amino acids (74-95), when preincubated with gp120, blocked its subsequent binding to CEM cells by 80%. A truncated form of this peptide (81-95), was found to be as efficient as the longer peptide (74-95) in inhibiting the binding of gp120 to CEM cells. The same peptide did not block the binding of OKT4A or Leu3A anti-CD4 monoclonal antibodies, which were previously shown to block HIV-I binding to CD4. The peptides were also tested for their ability to block HIV-I infection of a T cell line in vitro. Only CD4 peptide (74-95) and the shorter fragment (81-95) succeeded in protecting T cells against infection with different HIV-I strains. All the other peptides examined had no effect on gp120 binding to CEM cells and did not block syncytia formation. Goat polyclonal antibodies against the CD4 peptide (74-95) gave modest interference of gp120 binding to CEM cells. These data suggest that the CD4 region (74-95) participates in the CD4-mediated binding and/or internalization of HIV-I virion.     

Epitope mapping of a chimeric CD137 mAb: a necessary step for assessing the biologic relevance of non‐human primate models

Antibody based manipulation of the CD137 (4‐1BB) co‐signaling pathway is an attractive option for the treatment of cancer and autoimmune disease. We developed a chimeric anti‐human CD137 monoclonal antibody (GG) and characterized its function. As a component of planned preclinical studies, we evaluated the binding of GG to activated peripheral blood mononuclear cells (PBMCs) from cynomolgus macaque and baboon against human. Interestingly, GG only recognized human CD137, while a commercial anti‐CD137 mAb (4B4‐1), recognized activated PBMCs from both human and non‐human primates (NHP). Subsequent analysis revealed that the amino acid sequence of CD137 is largely conserved between primate species (～95% identical), with the extracellular domain differing by only 9–10 amino acids. Based on these data, we generated mutant constructs in the extracellular domain, replacing NHP with human CD137 sequences, and identified 3 amino acids critical for GG binding. These residues are likely part of a conformational epitope, as a peptide spanning this region is unable to block mAb binding. These data demonstrate that subtle sequence variations of defined co‐stimulatory molecules amongst primate species can be employed as a strategy for mapping residues necessary for antibody binding to conformational epitopes. Copyright © 2009 John Wiley & Sons, Ltd.     

Circular dichroism studies suggest that TAR RNA changes conformation upon specific binding of arginine or guanidine.

Short basic peptides from the HIV Tat protein bind specifically to a bulge region in TAR RNA, with a single arginine residue providing the only sequence-specific contact. The free amino acid arginine also binds specifically to TAR. Previous circular dichroism (CD) experiments suggested that peptide binding induces a conformational change in TAR. Here we confirm this observation using single arginine-containing peptides and show that arginine or guanidine binding also induces a conformational change in TAR. A peptide containing a single arginine within a stretch of histidines (CYHHHRHHHHHA) shows pH-dependent binding and a corresponding change in TAR conformation, as detected by a decrease in the CD signal at 265 nm. Arginine and guanidine, which bind to TAR with apparent Kd's of approximately 1.5 mM, induce similar CD changes. In contrast, lysine, which does not bind specifically to TAR, has no effect. Mutants of TAR that abolish specific binding (a U-->C substitution in the three-nucleotide bulge, a deletion of the bulge, or an A-U to U-A base pair change above the bulge) show no change in the CD signal upon binding of peptides, arginine, or guanidine. The results suggest that binding of a single guanidinium group to a specific site in TAR induces a change in RNA conformation.     

Characterization by NMR and molecular modeling of the binding of polyisoprenols and polyisoprenyl recognition sequence peptides: 3D structure of the complexes reveals sites of specific interactions

The objective of these studies was to test the hypothesis that proteins that contain potential polyisoprenyl recognition sequences (PIRSs) in their transmembrane-spanning domain can bind to the polyisoprenyl (PI) glycosyl carrier lipids undecaprenyl phosphate (C55-P) and dolichyl phosphate (C95-P). A number of prokaryotic and eukaryotic glycosyltransferases that utilize PI coenzymes contain a conserved PIRS postulated to be the active PI binding domain. To study this problem, we first determined the 3D structure of a PIRS peptide, NeuE, by homonuclear 2D 1H-nuclear magnetic resonance (NMR) spectroscopy. Experimentally generated distance constraints derived from nuclear Overhauser enhancement and torsion angle constraints derived from coupling constants were used for restrained molecular dynamics and energy minimization calculations. Molecular models of the NeuE peptide were built based on calculations of energy minimization using the DGII program NMRchitect. 3D models of dolichol (C95) and C95-P were built based on our 2D 1H-NMR nuclear Overhauser enhancement spectroscopy (NOESY) results and refined by energy minimization with respect to all atoms using the AMBER (assisted modeling with energy refinements) force field. Our energy minimization studies were carried out on a conformational model of dolichol that was originally derived from small-angle X-ray scattering and molecular mechanics methods. These results revealed that the PIs are conformationally nearly identical tripartite molecules, with their three domains arranged in a coiled, helical structure. Analyses of the intermolecular cross-peaks in the 2D NOESY spectra of PIRS peptides in the presence of PIs confirmed a highly specific interaction and identified key contact amino acids in the NeuE peptide that constituted a binding motif for interacting with the PIs. These studies also showed that subtle conformational changes occurred within both the PIs and the NeuE peptide after binding. 3D structures of the resulting molecular complexes revealed that each PI could bind more than one PIRS peptide. These studies thus represent the first evidence for a direct physical interaction between specific contact amino acids in the PIRS peptides and the PIs and supports the hypothesis of a bifunctional role for the PIs. The central idea is that these superlipids may serve as a structural scaffold to organize and stabilize in functional domains PIRS-containing proteins within multiglycosyltransferase complexes that participate in biosynthetic and translocation processes.     

Intensive Mutagenesis of the Nisin Hinge Leads to the Rational Design of Enhanced Derivatives

Nisin A is the most extensively studied lantibiotic and has been used as a preservative by the food industry since 1953. This 34 amino acid peptide contains three dehydrated amino acids and five thioether rings. These rings, resulting from one lanthionine and four methyllanthionine bridges, confer the peptide with its unique structure. Nisin A has two mechanisms of action, with the N-terminal domain of the peptide inhibiting cell wall synthesis through lipid II binding and the C-terminal domain responsible for pore-formation. The focus of this study is the three amino acid ‘hinge’ region (N 20, M 21 and K 22) which separates these two domains and allows for conformational flexibility. As all lantibiotics are gene encoded, novel variants can be generated through manipulation of the corresponding gene. A number of derivatives in which the hinge region was altered have previously been shown to possess enhanced antimicrobial activity. Here we take this approach further by employing simultaneous, indiscriminate site-saturation mutagenesis of all three hinge residues to create a novel bank of nisin derivative producers. Screening of this bank revealed that producers of peptides with hinge regions consisting of AAK, NAI and SLS displayed enhanced bioactivity against a variety of targets. These and other results suggested a preference for small, chiral amino acids within the hinge region, leading to the design and creation of producers of peptides with hinges consisting of AAA and SAA. These producers, and the corresponding peptides, exhibited enhanced bioactivity against Lactococcus lactis HP, Streptococcus agalactiae ATCC 13813, Mycobacterium smegmatis MC2155 and Staphylococcus aureus RF122 and thus represent the first example of nisin derivatives that possess enhanced activity as a consequence of rational design.     

Metadherin peptides containing CD4+ and CD8+ T cell epitopes as a therapeutic vaccine candidate against cancer

The concept of peptide‐based vaccines against cancer has made noteworthy progress. Metadherin (MTDH) overexpression and its role in the development of diverse cancers make it an attractive target for cancer immunotherapy. In the current study, six different T cell epitope prediction tools were run to identify MTDH peptides with multiple immunogenic regions. Further, molecular docking was performed to assess HLA‐peptide binding interactions. Nine and eleven peptides fragments containing multiple CD8 ⁺ and CD4 ⁺ T‐cell epitopes, ranging from 9 to 20 amino acids, respectively, were obtained using a consensus immunoinformatics approach. The three peptides that were finally identified as having overlapping CD4 ⁺ and CD8 ⁺ T‐ cell epitopes are ARLREMLSVGLGFLRTELG, FLLGYGWAAACAGAR, YIDDEWSGLNGLSSADP. These peptides were found to not only have multiple T cell epitopes but also to have binding affinity with wide HLA molecules. A molecular docking study revealed that the predicted immunogenic peptides (with single or multiple T cell epitopes) of MTDH have comparable binding energies with naturally bound peptides for both HLA classes I and II. Thus, these peptides have the potential to induce immune responses that could be considered for developing synthetic peptide vaccines against multiple cancers.     

The cell adhesion and proliferation activities of a peptide derived from human tenascin-C are dependent on two Ile residues

A tenascin-C derived peptide (TNIIIA2 peptide, 1) stimulated β1 integrin-mediated cell adhesion via binding to syndecan-4. Ala-substituted peptides were synthesized to understand the structure-activity relationship. Peptides in which basic amino acids were substituted showed reduced cell adhesion activity, but their proliferation activities were similar to or higher than those mediated by peptide 1. In contrast, peptides in which the Ile residues of peptide 1 were replaced were inactive, indicating that the Ile residues are critical for the peptide's activity. CD analysis suggested that the Ile residues are necessary for the formation of a specific conformation required for binding to syndecan-4.