Effect of molecular conformations on the adsorption behavior of gold-binding peptides

Despite extensive recent reports on combinatorially selected inorganic-binding peptides and their bionanotechnological utility as synthesizers and molecular linkers, there is still only limited knowledge about the molecular mechanisms of peptide binding to solid surfaces. There is, therefore, much work that needs to be carried out in terms of both the fundamentals of solid-binding kinetics of peptides and the effects of peptide primary and secondary structures on their recognition and binding to solid materials. Here we discuss the effects of constraints imposed on FliTrx-selected gold-binding peptide molecular structures upon their quantitative gold-binding affinity. We first selected two novel gold-binding peptide (AuBP) sequences using a FliTrx random peptide display library. These were, then, synthesized in two different forms: cyclic (c), reproducing the original FliTrx gold-binding sequence as displayed on bacterial cells, and linear (l) dodecapeptide gold-binding sequences. All four gold-binding peptides were then analyzed for their adsorption behavior using surface plasmon resonance spectroscopy. The peptides exhibit a range of binding affinities to and adsorption kinetics on gold surfaces, with the equilibrium constant, Keq, varying from 2.5x10(6) to 13.5x10(6) M(-1). Both circular dichroism and molecular mechanics/energy minimization studies reveal that each of the four peptides has various degrees of random coil and polyproline type II molecular conformations in solution. We found that AuBP1 retained its molecular conformation in both the c- and l-forms, and this is reflected in having similar adsorption behavior. On the other hand, the c- and l-forms of AuBP2 have different molecular structures, leading to differences in their gold-binding affinities.     

Adsorption kinetics of an engineered gold binding Peptide by surface plasmon resonance spectroscopy and a quartz crystal microbalance

The adsorption kinetics of an engineered gold binding peptide on gold surface was studied by using both quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) spectroscopy systems. The gold binding peptide was originally selected as a 14-amino acid sequence by cell surface display and then engineered to have a 3-repeat form (3R-GBP1) with improved binding characteristics. Both sets of adsorption data for 3R-GBP1 were fit to Langmuir models to extract kinetics and thermodynamics parameters. In SPR, the adsorption onto the surface shows a biexponential behavior and this is explained as the effect of bimodal surface topology of the polycrystalline gold substrate on 3R-GBP1 binding. Depending on the concentration of the peptide, a preferential adsorption on the surface takes place with different energy levels. The kinetic parameters (e.g., K(eq) approximately 10(7) M(-1)) and the binding energy (approximately -8.0 kcal/mol) are comparable to synthetic-based self-assembled monolayers. The results demonstrate the potential utilization of genetically engineered inorganic surface-specific peptides as molecular substrates due to their binding specificity, stability, and functionality in an aqueous-based environment.     

Chemistry of aqueous silica nanoparticle surfaces and the mechanism of selective peptide adsorption

Control over selective recognition of biomolecules on inorganic nanoparticles is a major challenge for the synthesis of new catalysts, functional carriers for therapeutics, and assembly of renewable biobased materials. We found low sequence similarity among sequences of peptides strongly attracted to amorphous silica nanoparticles of various size (15-450 nm) using combinatorial phage display methods. Characterization of the surface by acid base titrations and zeta potential measurements revealed that the acidity of the silica particles increased with larger particle size, corresponding to between 5% and 20% ionization of silanol groups at pH 7. The wide range of surface ionization results in the attraction of increasingly basic peptides to increasingly acidic nanoparticles, along with major changes in the aqueous interfacial layer as seen in molecular dynamics simulation. We identified the mechanism of peptide adsorption using binding assays, zeta potential measurements, IR spectra, and molecular simulations of the purified peptides (without phage) in contact with uniformly sized silica particles. Positively charged peptides are strongly attracted to anionic silica surfaces by ion pairing of protonated N-termini, Lys side chains, and Arg side chains with negatively charged siloxide groups. Further, attraction of the peptides to the surface involves hydrogen bonds between polar groups in the peptide with silanol and siloxide groups on the silica surface, as well as ion-dipole, dipole-dipole, and van-der-Waals interactions. Electrostatic attraction between peptides and particle surfaces is supported by neutralization of zeta potentials, an inverse correlation between the required peptide concentration for measurable adsorption and the peptide pI, and proximity of cationic groups to the surface in the computation. The importance of hydrogen bonds and polar interactions is supported by adsorption of noncationic peptides containing Ser, His, and Asp residues, including the formation of multilayers. We also demonstrate tuning of interfacial interactions using mutant peptides with an excellent correlation between adsorption measurements, zeta potentials, computed adsorption energies, and the proposed binding mechanism. Follow-on questions about the relation between peptide adsorption on silica nanoparticles and mineralization of silica from peptide-stabilized precursors are raised.     

Tuning secondary structure and self-assembly of amphiphilic peptides

Self-assembly is one of nature's mechanisms by which higher order structures are obtained. Two of the main driving forces for self-assembly, hydrophobic interactions and hydrogen bonding, are both present within amphiphilic peptides. Here, it is demonstrated how the intricately interconnected folding and assembly behavior of an N-terminally acylated peptide, with the sequence GANPNAAG, has been tuned by varying its hydrophobic tail and thermal history. The change in interplay between hydrophobic forces and peptide folding allowed the occurrence of different types of aggregation, from soluble peptides with a random coil conformation to aggregated peptides arranged in a beta-sheet assembly, which form helically twisted bilayer ribbons.     

Protein ligand design: from phage display to synthetic protein epitope mimetics in human antibody Fc-binding peptidomimetics

Phage display is a powerful method for selecting peptides with novel binding functions. Synthetic peptidomimetic chemistry is a powerful tool for creating structural diversity in ligands as a means to establish structure-activity relationships. Here we illustrate a method of bridging these two methodologies, by starting with a disulfide bridged phage display peptide which binds a human antibody Fc fragment (Delano et al. Science 2000, 287, 1279) and creating a backbone cyclic beta-hairpin peptidomimetic with 80-fold higher affinity for the Fc domain. The peptidomimetic is shown to adopt a well-defined beta-hairpin conformation in aqueous solution, with a bulge in one beta-strand, as seen in the crystal structure of the phage peptide bound to the Fc domain. The higher binding affinity of the peptidomimetic presumably reflects the effect of constraining the free ligand into the conformation required for binding, thus highlighting in this example the influence that ligand flexibility has on the binding energy. Since phage display peptides against a wide variety of different proteins are now accessible, this approach to synthetic ligand design might be applied to many other medicinally and biotechnologically interesting target proteins.     

具有双活性序列的新型抗菌肽的设计及性质

设计合成了具有2个活性序列的线性和环状多肽及具有单个活性序列的短链多肽, 研究了它们的杀菌活性、 细胞毒性及溶血性. 结果表明, 线性肽和环状肽的杀菌活性高于短链肽. 利用计算模拟的方法计算了多肽与细菌细胞膜中一种重要的成分磷脂酰甘油(DMPG)的结合能. 结果表明, 多肽-DMPG的结合能与多肽的杀菌活性具有较高的相关性, 线性和环状多肽与DMPG的结合能大于短链肽. 线性和环状多肽均含有2个活性序列, 可提供多个荷正电氨基酸与荷负电的磷脂结合, 结合能较大, 杀菌活性较强. 采用模拟生物膜对其中几条多肽的作用机理进行了初步研究. 结果表明, 该类多肽有可能使正常哺乳动物细胞的细胞膜产生孔洞; 而对于细菌细胞膜, 多肽并未在膜上产生明显孔洞, 而是引起了细菌细胞膜的聚集.     

Design of peptide that recognizes double-stranded DNA.

A novel molecular tool for double-stranded (ds) DNA detection using synthetic peptide is described. The peptide was designed based on the DNA binding domain of the lambda phage CRO repressor (CRO). The designed peptides contain helix-turn-helix (HTH), which is DNA binding motif. A cyclic peptide and a mutant peptide based on CRO were also designed, and the resulting affinity for dsDNA was increased. Furthermore, native amino acids of the peptide were replaced with arginine to increase the affinity for dsDNA. The affinity of these peptides for DNA binding was assessed by surface plasmon resonance (SPR) technique.     

新型抗菌肽的设计、活性研究及与磷脂相互作用的计算模拟

设计合成了多个具有2个活性序列的线性和环状多肽及具有单个活性序列的短链多肽, 研究了它们的杀菌活性, 发现其杀菌活性顺序为长链肽>环状肽>短链肽, 特别是线性的Linear-KT和Linear-KS对多种革兰氏阴性菌和阳性菌均具有较高的杀菌活性. 采用MTT法考察了Linear-KT和Linear-KS对正常细胞的毒性, 其中Linear-KS表现出较低的细胞毒性, 优于阳性对照多粘菌素B. 利用计算模拟的方法计算了多肽与细菌细胞膜中磷脂酰甘油(DMPG)的相互作用. 结果表明, 多肽和DMPG的结合能也表现出长链肽>环状肽>短链肽的规律, 特别是Linear-KT和Linear-KS具有较高的结合能. 长链肽含有2个活性序列, 可提供多个荷正电的氨基酸与荷负电的磷脂结合, 结合能较大, 杀菌活性较强. 同时, 柔性的结构及Linear-KT和Linear-KS中丝氨酸和苏氨酸的β碳上的羟基可与磷脂上的羰基形成多个氢键, 进一步增大了结合能. 计算模拟的方法为抗菌肽的杀菌活性从理论上提供了一定的依据.     

某些环肽及其相应线型肽在不同溶剂中构象的CD谱研究

We used CD spectroscopy to study the conformations of three cyclic peptides (CP10E: cyclo[Glu(OBz1)-Pro-Gly-Glu(OBzl)-Gly]2, CP10K: cyclo[Lys(Z)-Pro-Gly-Lys(Z)-Gly]2, CP12K: cyclo[Phe-Lys(Z)-Pro-Gly-Lys(Z)-Gly]2 and their correspondent linear peptides (LP10E: Boc-[Glu(OBzl)-Pro-Gly-Glu(OBzl)-Gly]2-OPac, LP10K: Boc-[Lys(Z)-Pro-Lys(Z)-Pro]2-OMe, LP 12K: Bao- [-Lys(Z)-Pro-Gly-Lys(Z)-Gly]2- OMe) in three solvents of different polarity (chloroform, acetonitrile, 2,2,2-triliuroethanol), and it was found that all of linear and cyclicpeptides exists asγ-turn conformation in chloroform, however, in TFE＆ CH3CN solutions, the three linear peptides are inβ Ⅱ-turn conformations. CP10E isβI-turn conformation, CP10K ＆CP12K exists in more than one types of turn conformations. On the basis of our experiments, it was concluded: 1) In the presence of conformational constrained amino acids short linear peptides form obvious secondary structure; 2)The solvent polarity has influence on the peptide conformation and this influence on linear peptides is greater than that on cyclic peptides; 3)The backbone of cyclic peptide has constraint effect on its conformation and makes the secondary structure of cyclic peptide different from that of its relative linear peptide. This information might give some cules in the design of bioactive peptides with different receptor selectivity.     

Isolation of ZnO-binding 12-mer peptides and determination of their binding epitopes by NMR spectroscopy

Inorganic-binding peptides are in the focus of research fields such as materials science, nanotechnology, and biotechnology. Applications concern surface functionalization by the specific coupling to inorganic target substrates, the binding of soluble molecules for sensing applications, or biomineralization approaches for the controlled formation of inorganic materials. The specific molecular recognition of inorganic surfaces by peptides is of major importance for such applications. Zinc oxide (ZnO) is an important semiconductor material which is applied in various devices. In this study the molecular fundamentals for a ZnO-binding epitope was determined. 12-mer peptides, which specifically bind to the zinc- or/and the oxygen-terminated sides of single-crystalline ZnO (0001) and (000-1) substrates, were selected from a random peptide library using the phage display technique. For two ZnO-binding peptides the mandatory amino acid residues, which are of crucial importance for the specific binding were determined with a label-free nuclear magnetic resonance (NMR) approach. NMR spectroscopy allows the identification of pH dependent interaction sites on the atomic level of 12-mer peptides and ZnO nanoparticles. Here, ionic and polar interaction forces were determined. For the oxygen-terminated side the consensus peptide-binding sequence (HSXXH) was predicted in silico and confirmed by the NMR approach.     

High-throughput sequence determination of cyclic peptide library members by partial Edman degradation/mass spectrometry

Cyclic peptides provide attractive lead compounds for drug discovery and excellent molecular probes in biomedical research. Large combinatorial libraries of cyclic peptides can now be routinely synthesized by the split-and-pool method and screened against biological targets. However, post-screening sequence determination of hit peptides has been problematic. In this report, a high-throughput method for the sequence determination of cyclic peptide library members has been developed. TentaGel microbeads (90 mum) were spatially segregated into outer and inner layers; cyclic peptides were displayed on the bead surface, whereas the inner core of each bead contained the corresponding linear peptide as the encoding sequence. After screening of the cyclic peptide library against a macromolecular target, the identity of hit peptides was determined by sequencing the linear encoding peptides inside the bead using a partial Edman degradation/mass spectrometry method. On-bead screening of an octapeptide library (theoretical diversity of 160 000) identified cyclic peptides that bind to streptavidin. A 400-member library of tyrocidine A analogues was synthesized on TentaGel macrobeads and solution-phase screening of the library directly against bacterial cells identified a tyrocidine analogue of improved antibacterial activity. Our results demonstrate that the new method for cyclic peptide sequence determination is reliable, operationally simple, rapid, and inexpensive and should greatly expand the utility of cyclic peptides in biomedical research.     

Biological identification of peptides that specifically bind to poly(phenylene vinylene) surfaces: recognition of the branched or linear structure of the conjugated polymer

Peptides that bind to poly(phenylene vinylene) (PPV) were identified by the phage display method. Aromatic amino acids were enriched in these peptide sequences, suggesting that a π-π interaction is the key interaction between the peptides and PPV. The surface plasmon resonance (SPR) experiments using chemically synthesized peptides demonstrated that the Hyp01 peptide, with the sequence His-Thr-Asp-Trp-Arg-Leu-Gly-Thr-Trp-His-His-Ser, showed an affinity constant (7.7 × 10(5) M(-1)) for the target, hyperbranched PPV (hypPPV) film. This value is 15-fold greater than its affinity for linear PPV (linPPV). In contrast, the peptide screened for linPPV (Lin01) showed the reverse specificity for linPPV. These results suggested that the Hyp01 and Lin01 peptides selectively recognized the linear or branched structure of PPVs. The Ala-scanning experiment, circular dichroism (CD) spectrometry, and molecular modeling of the Hyp01 peptide indicated that adequate location of two Trp residues by forming the polyproline type II (P(II)) helical conformation allowed the peptide to specifically interact with hypPPV.     

Biological selection of peptides for poly(l-lactide) substrates

Short peptides that recognize the alpha form of poly( l-lactide) (PLLA) crystalline films were identified from a phage-displayed peptide library. An enzyme-linked immunosorbent assay (ELISA) revealed that the apparent binding constants of the phage clones for the alpha form of PLLA were greater than those of the unselected phage library. The specificity index for the alpha form of PLLA referred to a structurally similar atactic poly(methyl methacrylate) (at-PMMA), supporting the alpha form of PLLA specific binding of the selected phage. Amino acid residues with proton-donor lateral groups and hydrophobic alkyl groups were relatively enriched in a sequence of heptapeptides on the specific phage clones, thereby suggesting the presence of hydrogen bonding as well as hydrophobic interactions between the alpha form of PLLA and the peptides. Surface plasmon resonance (SPR) analysis revealed that the binding constant of the freed c22 heptapeptide (Gln-Leu-Met-His-Asp-Tyr-Arg) for the alpha form of PLLA was greater than those for reference at-PMMA, amorphous PLLA, and the beta form of PLLA. It was found that c22 peptide can recognize slight differences in PLLA polymorphs such as a crystalline state and an arrangement of PLLA functional groups.     

Membrane binding and structure of de novo designed alpha-helical cationic coiled-coil-forming peptides.

We introduce a de novo designed peptide model system that enables the systematic study of 1) the role of a membrane environment in coiled-coil peptide folding, 2) the impact of different domains of an alpha-helical coiled-coil heptad repeat on the interaction with membranes, and 3) the dynamics of coiled-coil peptide-membrane interactions depending on environmental conditions. Starting from an ideal alpha-helical coiled-coil peptide sequence, several positively charged analogues were designed that exhibit a high propensity toward negatively charged lipid membranes. Furthermore, these peptides differ in their ability to form a stable alpha-helical coiled-coil structure. The influence of a membrane environment on peptide folding is studied. All positively charged peptides show strong interactions with negatively charged membranes. This interaction induces an alpha-helical structure of the former random-coil peptides, as revealed by circular dichroism measurements. Furthermore, vesicle aggregation is induced by a coiled-coil interaction of vesicle-bound peptides. Dynamic light scattering experiments show that the strength of vesicle aggregation increases with the peptide's intrinsic ability to form a stable alpha-helical coiled coil. Thus, the peptide variant equipped with the strongest inter- and intra-helical coiled-coil interactions shows the strongest effect on vesicle aggregation. The secondary structure of this peptide in the membrane-bound state was studied as well as its effect on the phospholipids. Peptide conformation within the peptide-lipid aggregates was analyzed by (13)C cross-polarization magic-angle spinning NMR experiments. A uniformly (13)C- and (15)N-labeled Leu residue was introduced at position 12 of the peptide chain. The (13)C chemical shift and torsion angle measurements support the finding of an alpha-helical structure of the peptide in its membrane-bound state. Neither membrane leakage nor fusion was observed upon peptide binding, which is unusual for amphiphatic peptide structures. Our results lay the foundation for a systematic study of the influence of the alpha-helical coiled-coil folding motif in membrane-active events on a molecular level.     

N-methylated cyclic pentaalanine peptides as template structures

The N-methylation of cyclic peptides can be used to modify the activity and/or selectivity of biologically active peptides. As N-methylation introduces different flexibility and lipophilicity, it can also improve the bioavailability (the ADMET profile). To search for conformationally constrained cyclic peptides, a library of 30 different N-methylated peptides with the basic sequence cyclo(-D-Ala-L-Ala4-) was synthesized. Based on the NMR analysis, seven of these peptides exhibited single conformations (>98%). The structural features of these peptides were determined by a combination of NMR and distance geometry and then further refined by molecular dynamics simulations in an explicit DMSO solvent box. The structures provided from these efforts can now serve as templates for the rational design of cyclic pentapeptides with a distinct backbone conformation or for "spatial screening" to explore the bioactive conformation of medically important peptide systems.     

A Genetically Encoded,Phage‐Displayed Cyclic‐Peptide Library

Superior to linear peptides in biological activities, cyclic peptides are considered to have great potential as therapeutic agents. To identify cyclic‐peptide ligands for therapeutic targets, phage‐displayed peptide libraries in which cyclization is achieved by the covalent conjugation of cysteines have been widely used. To resolve drawbacks related to cysteine conjugation, we have invented a phage‐display technique in which its displayed peptides are cyclized through a proximity‐driven Michael addition reaction between a cysteine and an amber‐codon‐encoded N^?‐acryloyl‐lysine (AcrK). Using a randomized 6‐mer library in which peptides were cyclized at two ends through a cysteine–AcrK linker, we demonstrated the successful selection of potent ligands for TEV protease and HDAC8. All selected cyclic peptide ligands showed 4‐ to 6‐fold stronger affinity to their protein targets than their linear counterparts. We believe this approach will find broad applications in drug discovery.     

A Genetically Encoded,Phage‐Displayed Cyclic‐Peptide Library

Superior to linear peptides in biological activities, cyclic peptides are considered to have great potential as therapeutic agents. To identify cyclic‐peptide ligands for therapeutic targets, phage‐displayed peptide libraries in which cyclization is achieved by the covalent conjugation of cysteines have been widely used. To resolve drawbacks related to cysteine conjugation, we have invented a phage‐display technique in which its displayed peptides are cyclized through a proximity‐driven Michael addition reaction between a cysteine and an amber‐codon‐encoded N^?‐acryloyl‐lysine (AcrK). Using a randomized 6‐mer library in which peptides were cyclized at two ends through a cysteine–AcrK linker, we demonstrated the successful selection of potent ligands for TEV protease and HDAC8. All selected cyclic peptide ligands showed 4‐ to 6‐fold stronger affinity to their protein targets than their linear counterparts. We believe this approach will find broad applications in drug discovery.     

Phage-Display Based Discovery and Characterization of Peptide Ligands against WDR5

WD40 is a ubiquitous domain presented in at least 361 human proteins and acts as scaffold to form protein complexes. Among them, WDR5 protein is an important mediator in several protein complexes to exert its functions in histone modification and chromatin remodeling. Therefore, it was considered as a promising epigenetic target involving in anti-cancer drug development. In view of the protein–protein interaction nature of WDR5, we initialized a campaign to discover new peptide-mimic inhibitors of WDR5. In current study, we utilized the phage display technique and screened with a disulfide-based cyclic peptide phage library. Five rounds of biopanning were performed and isolated clones were sequenced. By analyzing the sequences, total five peptides were synthesized for binding assay. The four peptides are shown to have the moderate binding affinity. Finally, the detailed binding interactions were revealed by solving a WDR5-peptide cocrystal structure.     

A Cysteine-Directed Proximity-Driven Crosslinking Method for Native Peptide Bicyclization

Efficient and site-specific modification of native peptides and proteins is desirable for synthesizing antibody-drug conjugates as well as for constructing chemically modified peptide libraries using genetically encoded platforms such as phage display. In particular, there is much interest in efficient multicyclization of native peptides due to the appeals of multicyclic peptides as therapeutics. However, conventional approaches for multicyclic peptide synthesis require orthogonal protecting groups or non-proteinogenic clickable handles. Herein, we report a cysteine-directed proximity-driven strategy for the constructing bicyclic peptides from simple natural peptide precursors. This linear to bicycle transformation initiates with rapid cysteine labeling, which then triggers proximity-driven amine-selective cyclization. This bicyclization proceeds rapidly under physiologic conditions, yielding bicyclic peptides with a Cys-Lys-Cys, Lys-Cys-Lys or N-terminus-Cys-Cys stapling pattern. We demonstrate the utility and power of this strategy by constructing bicyclic peptides fused to proteins as well as to the M13 phage, paving the way to phage display of novel bicyclic peptide libraries.     

Does adsorption at hydroxyapatite surfaces induce peptide folding? Insights from large-scale B3LYP calculations

Large-scale periodic quantum mechanical calculations (509 atoms, 7852 atomic orbitals) based on the hybrid B3LYP functional focused on the peptide folding induced by the adsorption on the (001) and (010) hydroxyapatite (HA) surfaces give interesting insights on the role of specific interactions between surface sites and the peptide, which stabilize the helix conformation over the "native" random coil ones for in silico designed model peptides. The two peptides were derived from the 12-Gly oligomer, with one (P1, C-tGGKGGGGGGEGGN-t) and two (P2, C-tGGKGGKEGGEGGN-t) glutamic acid (E) and lysine (K) residue mutations. The most stable gas-phase "native" conformation for both peptides resulted in a random coil (RC) structure, with the helix (H) conformation being ≈100 kJ mol(-1) higher in free energy. The two peptide conformations interact with the HA (001) and (010) surfaces by C═O groups via Ca(2+) ions, by hydrogen bond between NH(2) groups and the basic PO(4)(3-) groups and by a relevant fraction due to dispersion forces. Peptide adsorption was studied on the dry (001) surface, the wet one envisaging 2 H(2)O per surface Ca(2+) and, on the latter, also considering the adsorption of microsolvated peptides with 4 H(2)O molecules located at sites responsible of the interaction with the surface. The P1 mutant does prefer to be adsorbed as a random coil by ≈160 kJ/mol, whereas the reverse is computed for P2, preferring the helix conformation by ≈50 kJ/mol. Adsorption as helix of both P1 and P2 mutants brings about proton transfer toward the HA surfaces with a large charge transfer component to the interaction energy.