Multiple-injection affinity capillary electrophoresis to examine binding constants between glycopeptide antibiotics and peptides

Multiple-injection affinity capillary electrophoresis (MIACE) was used to determine binding constants (K(b)) between vancomycin, ristocetin, and teicoplanin from Streptomyces orientalis, Nocardia lurida, and Actinoplanes teichomyceticus, respectively, and fluorenylmethoxycarbonyl (Fmoc)-(Gly, Ala, Val, and Phe)-D-Ala-D-Ala peptides. In this technique, separate plugs of sample containing non-interacting standards, peptide one, buffer, and peptide two, were injected into the capillary column and electrophoresed. Peptides migrate through the column at similar electrophoretic mobilities but remain as distinct zones due to the buffer plug between peptides. The electrophoresis is then carried out in an increasing concentration of antibiotic in the running buffer. Continued electrophoresis results in a shift in the migration time of the peptides upon binding to the antibiotic. Analysis of the change in the relative migration time ratio (RMTR) of the resultant complexes relative to the non-interacting standards, as a function of the concentration of antibiotic yields a value for K(b). MIACE is a versatile technique that can be used to measure affinity constants between ligands of similar relative molecular mass and charge without the need of separate binding experiments. The findings described, herein, demonstrate the advantages of using MIACE to estimate binding parameters between ligands and receptors.     

Design, optimization and evaluation of specific affinity adsorbent for oligopeptides

A major challenge in the development of affinity adsorbents is the design of specific adsorbents for target molecules. In this paper, a two-step strategy was used to design a specific adsorbent for oligopeptides. Based on the structural characteristics of target peptide DFLAE (DE5), the affinity ligand CDenHis bearing hydrophobic inclusion and electrostatic interaction sites was prepared by grafting histidine onto β-cyclodextrin (CD) using ethylenediamine; ligands with single hydrophobic inclusion or electrostatic interaction sites (CDen and HisOMe) were used as reference ligands. Results indicated that the binding affinity (K(a)) of CDenHis with DE5 was 6.23×10(4)M(-1), 23- and 61-fold higher than that of CDen and HisOMe, respectively. Computer simulations were used to further optimize the steric configuration of CDenHis. It was found that the optimized ligand CDdnHis exhibited a much improved binding affinity for DE5 (K(a)=1.02×10(5)M(-1)). Moreover, the corresponding adsorbent A-CDdnHis not only showed much better adsorption ability compared with A-CDenHis, but also excellent adsorption specificity for DE5-containing peptides. Kinetic analysis and adsorption mechanism studies suggested that the configuration matching of CDdnHis with DE5 and the cooperation of multiple interactions led to the fast and selective adsorption of DE5-containing peptides to A-CDdnHis.     

Identification and refinement of a peptide affinity ligand with unique specificity for a monoclonal anti-tenascin-C antibody by screening of a phage display library

Using phage display technology, a 22-mer peptide was selected as a ligand with unique specificity for the murine monoclonal ST2146 antibody that recognizes the EGF repeats region of the human tumor-associated antigen tenascin-C. This peptide, synthesized in an 8-branched form to enhance its binding properties, is useful in replacing the native antigen in the affinity and immunoreactivity characterization of the ST2146 antibody and its biotinylated derivatives. Affinity resins, prepared by immobilizing the mimotope or its shorter 10-mer binding unit on a chromatographic support, were able to capture ST2146 directly from the hybridoma supernatant, with antibody recovery and host cell protein (HCP) reduction similar to or better than protein A sorbent, a purity degree exceeding 95%, and full recovery of antibody activity. The affinity constants of both peptides, as determined by frontal analysis of broad-zone elution affinity chromatography and BiaCore measurements, were very similar and included in a range suitable for affinity ligands. Column capacity, determined by applying a large excess of purified ST2146 to 1 mL of column bed volume, was close to 50 mg/mL for both resins. These matrices retain their ST2146 binding properties after various treatments, including sanitization, thus indicating very high stability in terms of ligand leakage and degradation. Moreover, the short form shows higher enzymatic stability, thus proving more suitable as ligand for ST2146 affinity purification.     

Investigations of cyclophilin interactions with oligopeptides containing proline by affinity capillary electrophoresis

Affinity capillary electrophoresis using mobility-shift analysis was utilized to characterize the binding of peptide ligands to cyclophilins, which are members of the enzyme family of peptidyl-prolyl cis/trans isomerases. Peptides derived from the human immunodeficiency virus capsid protein p24 exhibited different affinities to the isoenzymes cyclophilin18 and cyclophilin20. For the interaction of the peptide hormone bradykinin with cyclophilin18, a dissociation constant of 1.4 +/- 0.1 mM was determined. Finally, the affinity of cyclophilin20 to peptides from a cellulose-bound peptide library scanning the sequence of Drosophila melanogaster protein cappuccino was investigated. The affinities of selected peptides to cyclophilin20 and a green fluorescent fusion protein with cyclophilin20 were compared.     

Dissecting the Engrailed homeodomain-DNA interaction by phage-displayed shotgun scanning

Phage-displayed alanine shotgun scanning was used to dissect contributions by engrailed homedomain (En-HD) residues 17 through 46, which indirectly influence recognition of DNA. The relative contributions of such indirect contacts, quantified by shotgun scanning, highlight previously unexplored En-HD residues. Two motifs dominate En-HD function in this region. First, two surface-exposed aromatic residues (F20 and Y25) bracket the hydrophobic core. Second, two sets of turn-forming residues are highlighted, including carboxamide-requiring residues E22/N23 and a leucine/isoleucine splint. The En-HD hydrophobic core exhibits a surprising degree of malleability, as demonstrated by homolog shotgun scanning. Most selectants from in vitro shotgun scanning mirror the consensus human homeodomain sequence. Thus, natural evolution and in vitro selection use similar selection criteria: affinity, specificity, and stability. However, homolog shotgun scanning identified mutations capable of improving the affinity and specificity of En-HD.     

Oriented immobilization of peptide ligands on solid supports.

A synthetic procedure was developed for the direct immobilization on preactivated affinity supports of peptidic ligands requiring free alpha-amino groups to recognize their targets properly. The peptidic ligand is assembled by solid-phase peptide synthesis on an octa-branched heptalysine core through a polyglycine spacer, similar to the method developed for the production of multiple antigenic peptides. After deblocking from the resin, peptide is dialysed, lyophylized and used directly for coupling to preactivated supports. Following immobilization, only a limited number of peptide chains are covalently linked to the solid phase, leaving the remainder facing the mobile phase and sufficiently spaced to interact properly. This procedure was applied successfully to the design, synthesis and oriented immobilization of a multimeric tripeptide ligand (Met-Tyr-Phe) for affinity purification of bovine neurophysin.     

Partial filling multiple injection affinity capillary electrophoresis (PFMIACE) to estimate binding constants of receptors to ligands

Partial filling multiple injection affinity capillary electrophoresis (PFMIACE) is used to determine binding constants between vancomycin (Van) from Streptomyces orientalis, teicoplanin (Teic) from Actinoplanes teicomyceticus and ristocetin (Rist) from Nocardia lurida to d-Ala-d-Ala terminus peptides and carbonic anhydrase B (CAB, E.C.4.2.1.1) to arylsulfonamides. Two variations of PFMIACE are described herein. In the first technique, the capillary is partially filled with ligand at increasing concentrations, a non-interacting standard, three or four separate plugs of receptor each separated by small plugs of buffer, a plug containing a second non-interacting standard, and then electrophoresed in buffer. Upon continued electrophoresis, equilibrium is established between the ligand and receptors causing a shift in the migration time of the receptors with respect to the non-interacting standards. This change in migration time is utilized for estimating multiple binding constants (K_b) for the same interaction. In the second technique, separate plugs of sample containing non-interacting standards, peptide one, buffer, and peptide two, were injected into the capillary column. The capillary is partially filled with a series of buffers containing an antibiotic at increasing concentrations and electrophoresed. Peptides migrate through the column at similar electrophoretic mobilities since their charge-to-mass ratios are approximately the same but remain as distinct zones due to the buffer plug between peptides. Upon electrophoresis, the plug of antibiotic flows into the peptide plugs affecting a shift in the migration time of the peptides with respect to the non-interacting standards occurs due to formation of the of the antibiotic-peptide complex. The shift in the migration time of the peptides upon binding to the antibiotic is used for the Scatchard analysis and measurement of a K_b. The PFMIACE technique expands the functionality and potential of ACE as an analytical tool to examine receptor-ligand interactions. In PFMIACE, a smaller amount of sample is required in the assay compared to both conventional ACE and MIACE. Furthermore, a wide array of data is obtained from a single experiment, thus, expediting the assay of biological species.     

Structural and ITC Characterization of Peptide-Protein Binding: Thermodynamic Consequences of Cyclization Constraints,a Case Study on Vascular Endothelial Growth Factor Ligands

Macrocyclization constraints are widely used in the design of protein ligands to stabilize their bioactive conformation and increase their affinities. However, the resulting changes in binding entropy can be puzzling and uncorrelated to affinity gains. Here, the thermodynamic (Isothermal Titration Calorimetry) and structural (X-ray, NMR and CD) analysis of a complete series of lactam-bridged peptide ligands of the vascular endothelial growth factor, and their unconstrained analogs are reported. It is shown that differences in thermodynamics arise mainly from the folding energy of the peptide upon binding. The systematic reduction in conformational entropy penalty due to helix pre-organization can be counterbalanced by an unfavorable vibrational entropy change if the constraints are too rigid. The gain in configurational entropy partially escapes the enthalpy/entropy compensation and leads to an improvement in affinity. The precision of the analytical ITC method makes this study a possible benchmark for constrained peptides optimization.     

Resolving Multiple Protein–Peptide Binding Events: Implication for HLA-DQ2 Mediated Antigen Presentation in Celiac Disease

Techniques that can effectively separate protein–peptide complexes from free peptides have shown great value in major histocompatibility complex (MHC)–peptide binding studies. However, most of the available techniques are limited to measuring the binding of a single peptide to an MHC molecule. As antigen presentation in vivo involves both endogenous ligands and exogenous antigens, the deconvolution of multiple binding events necessitates the implementation of a more powerful technique. Here we show that capillary electrophoresis coupled to fluorescence detection (CE–FL) can resolve multiple MHC–peptide binding events owing to its superior resolution and the ability to simultaneously monitor multiple emission channels. We utilized CE–FL to investigate competition and displacement of endogenous peptides by an immunogenic gluten peptide for binding to HLA-DQ2. Remarkably, this immunogenic peptide could displace CLIP peptides from the DQ2 binding site at neutral but not acidic pH. This unusual ability of the gluten peptide supports a direct loading mechanism of antigen presentation in extracellular environment, a property that could explain the antigenicity of dietary gluten in celiac disease.     

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.     

Contribution of individual zinc ligands to metal binding and peptide folding of zinc finger peptides

Little is known about the contribution of individual zinc-ligating amino acid residues for coupling between zinc binding and protein folding in zinc finger domains. To understand such roles of each zinc ligand, four zinc finger mutant peptides corresponding to the second zinc finger domain of Sp1 were synthesized. In the mutant peptides, glycine was substituted for one of four zinc ligands. Their metal binding and folding properties were spectroscopically characterized and compared to those of the native zinc finger peptide. In particular, the electronic charge-transfer and d-d bands of the Co(II)-substituted peptide complexes were used to examine the metal coordination number and geometry. Fluorescence emission studies revealed that the mutant peptides are capable of binding zinc despite removing one ligand. Circular dichroism results clearly showed the induction of an alpha-helix by zinc binding. In addition, the structures of certain mutant zinc finger peptides were simulated by molecular dynamics calculation. The information indicates that His23 and the hydrophobic core formed between the alpha-helix and the beta-sheet play an essential role in alpha-helix induction. This report demonstrates that each ligand does not contribute equally to alpha-helix formation and coordination geometry in the zinc finger peptide.     

Study on the degeneracy of antisense peptides using affinity chromatography

The degeneracy of antisense peptides was studied by high-performance affinity chromatography. A model sense peptide (AAAA) and its antisense peptides (CGGG, GGGG, RGGG, SGGG) were designed and synthesized according to the degeneracy of genetic codes. An affinity column with AAAA as the ligand was prepared. The affinity chromatographic behaviors of antisense peptides on the column were evaluated. The results indicated that model antisense peptides have clear retention on the immobilized AAAA affinity column. RGGG showed the strongest affinity interaction. Similar result was obtained from another experiment that Arg-substituted antisense peptide of fusion peptide (1-11) of influenza virus A was also shown the highest affinity binding to immobilized fusion peptide.     

A general technique to rank protein-ligand binding affinities and determine allosteric versus direct binding site competition in compound mixtures

To realize the full potential of combinatorial chemistry-based drug discovery, generic and efficient tools must be developed that apply the strengths of diversity-oriented chemical synthesis to the identification and optimization of lead compounds for disease-associated protein targets. We report an affinity selection-mass spectrometry (AS-MS) method for protein-ligand affinity ranking and the classification of ligands by binding site. The method incorporates the following steps: (1) an affinity selection stage, where protein-binding compounds are selected from pools of ligands in the presence of varying concentrations of a competitor ligand, (2) a first chromatography stage to separate unbound ligands from protein-ligand complexes, and (3) a second chromatography stage to dissociate the ligands from the complexes for identification and quantification by MS. The ability of the competitor ligand to displace a target-bound library member, as measured by MS, reveals the binding site classification and affinity ranking of the mixture components. The technique requires no radiolabel incorporation or direct biochemical assay, no modification or immobilization of the compounds or target protein, and all reaction components, including any buffers or cofactors required for protein stability, are free in solution. We demonstrate the method for several compounds of wide structural variety against representatives of the most important protein classes in contemporary drug discovery, including novel ATP-competitive and allosteric inhibitors of the Akt-1 (PKB) and Zap-70 kinases, and previously undisclosed antagonists of the M(2) muscarinic acetylcholine receptor, a G-protein coupled receptor (GPCR). The theoretical basis of the technique is analyzed mathematically, allowing quantitative estimation of binding affinities and, in the case of allosteric interaction, absolute determination of binding cooperativity. The method is readily applicable to high-throughput screening hit triage, combinatorial library-based affinity optimization, and developing structure-activity relationships among multiple ligands to a given receptor.     

Trypsin and affinity chromatography.

Affinity adsorbents for trypsin which were prepared by immobilizing product-type ligands, that is, peptides having C-terminal arginine, proved to be effective not only for preparative purposes but also for basic research on molecular recognition. The properties of the binding site of trypsin were revealed by chromatographic experiments. Quantitative analysis based on the theory of frontal affinity chromatography proved to be extremely effective. As an extension of the product-type ligands, peptide argininals were also used and information on the mechanism of action of these inhibitors was obtained. Anhydrotrypsin, which lost the hydroxyl group of Ser183, was found to gain increased binding ability for product-type compounds. This inactivated enzyme was also used as an immobilized ligand and the unique affinity adsorbent thus prepared proved to be extremely effective for the separation of peptides and recombinant proteins based on their C-terminal structures. High-performance affinity chromatography of trypsin and related enzymes using a polymer-based support was also developed.     

Identification of small molecule binding sites within proteins using phage display technology

Affinity selection of peptides displayed on phage particles was used as the basis for mapping molecular contacts between small molecule ligands and their protein targets. Analysis of the crystal structures of complexes between proteins and small molecule ligands revealed that virtually all ligands of molecular weight 300 Da or greater have a continuous binding epitope of 5 residues or more. This observation led to the development of a technique for binding site identification which involves statistical analysis of an affinity-selected set of peptides obtained by screening of libraries of random, phage-displayed peptides against small molecules attached to solid surfaces. A random sample of the selected peptides is sequenced and used as input for a similarity scanning program which calculates cumulative similarity scores along the length of the putative receptor. Regions of the protein sequence exhibiting the highest similarity with the selected peptides proved to have a high probability of being involved in ligand binding. This technique has been employed successfully to map the contact residues in multiple known targets of the anticancer drugs paclitaxel (Taxol), docetaxel (Taxotere) and 2-methoxyestradiol and the glycosaminoglycan hyaluronan, and to identify a novel paclitaxel receptor [1]. These data corroborate the observation that the binding properties of peptides displayed on the surface of phage particles can mimic the binding properties of peptides in naturally occurring proteins. It follows directly that structural context is relatively unimportant for determining the binding properties of these disordered peptides. This technique represents a novel, rapid, high resolution method for identifying potential ligand binding sites in the absence of three-dimensional information and has the potential to greatly enhance the speed of development of novel small molecule pharmaceuticals.     

Peptides that mimic glycosaminoglycans: high-affinity ligands for a hyaluronan binding domain.

BACKGROUND: Hyaluronan (HA) is a non-sulfated glycosaminoglycan (GAG) that promotes motility, adhesion, and proliferation in mammalian cells, as mediated by cell-surface HA receptors. We sought to identify non-carbohydrate ligands that would bind to and activate cell-surface HA receptors. Such analogs could have important therapeutic uses in the treatment of cancer, wound healing, and arthritis, since such ligands would be resistant to degradation by hyaluronidase (HAse). RESULTS: Peptide ligands that bind specifically to the recombinant HA binding domain (BD) of the receptor for hyaluronan-mediated motility (RHAMM) were obtained by screening two peptide libraries: (i) random 8-mers and (ii) biased 8-mers with alternating acidic side chains, i.e. XZXZXZXZ (X=all-L-amino acids except Cys, Lys, or Arg; Z=D-Asp, L-Asp, D-Glu, or L-Glu). Selectivity of the peptide ligands for the HABD was established by (i) detection of binding of biotin- or fluorescein-labeled peptides to immobilized proteins and (ii) fluorescence polarization of FITC-labeled peptides with the HABD in solution. HA competitively displaced binding of peptides to the HABD, while other GAGs were less effective competitors. The stereochemistry of four biased octapeptides was established by synthesis of the 16 stereoisomers of each peptide. Binding assays demonstrated a strong preference for alternating D and L configurations for the acidic residues, consistent with the calculated orientation of glucuronic acid moieties of HA. CONCLUSIONS: Two classes of HAse-resistant peptide mimetics of HA were identified with high affinity, HA-compatible binding to the RHAMM HABD. This demonstrated that non-HA ligands specific to a given HA binding protein could be engineered, permitting receptor-specific targeting.     

Capillary electrophoretic methods applied to the investigation of peptide complexes

This article gives an overview of the applications of capillary electrophoretic methods to investigate the non‐covalent interactions of peptides (peptide complexes) with variable middle‐ and high‐molecular‐mass receptors (ligands) as well as with small ions and molecules in the period 2007–2014. Different modes of capillary electrophoretic methods, such as mobility shift (vacancy) affinity capillary electrophoresis, multiple injection affinity capillary electrophoresis, partial filling affinity capillary electrophoresis, Hummel–Dryer method, vacancy peak method and (continuous) frontal analysis capillary electrophoresis, are briefly described and their applicability to determination of binding constants of peptide complexes is discussed. In addition, the detailed experimental conditions of individual applications and the values of binding constants of the particular peptide complexes are presented.     

On-column synthesis coupled to affinity capillary electrophoresis for the determination of binding constants of peptides to glycopeptide antibiotics

Binding constants of the glycopeptide antibiotics teicoplanin (Teic), ristocetin (Rist), and vancomycin (Van), and their derivatives to D-Ala-D-Ala terminus peptides were determined by on-column ligand and receptor synthesis coupled to affinity capillary electrophoresis (ACE) or partial filling ACE (PFACE). In the first technique, 9-fluorenylmethoxycarbonyl (Fmoc)-amino acid-D-Ala-D-Ala species are first synthesized using on-column techniques. The initial sample plug contains a D-Ala-D-Ala terminus peptide and two non-interacting standards. Plugs two and three contain solutions of Fmoc-amino acid-N-hydroxysuccinimide (NHS) ester and buffer, respectively. Upon electrophoresis, the initial D-Ala-D-Ala peptide reacts with the Fmoc-amino acid NHS ester yielding the Fmoc-amino acid D-Ala-D-Ala peptide. Continued electrophoresis results in the overlap of the glycopeptide in the running buffer and the plug of Fmoc-amino acid-D-Ala-D-Ala peptide and non-interacting markers. Subsequent analysis of the change in the electrophoretic mobility (mu) or relative migration time ratio (RMTR) of the peptide relative to the non-interacting standards, as a function of the concentration of the antibiotic, yields a value for the binding constant. In the second technique, derivatives of the glycopeptides Teic and Rist are first synthesized on-column before analysis by ACE or PFACE. After the column has been partially filled with increasing concentrations of D-Ala-D-Ala terminus peptides, a plug of buffer followed by two separate plugs of reagents are injected. The order of the reagent plugs containing the antibiotic and two non-interacting standards and the anhydride varies with the charge of the glycopeptide. Upon electrophoresis, the antibiotic reacts with the anhydride yielding a derivative of Teic or Rist. Continued electrophoresis results in the overlap of the derivatized antibiotic and the plug of D-Ala-D-Ala peptide. Analysis of the change in RMTR of the new glycopeptide relative to the non-interacting standards, as a function of the concentration of the D-Ala-D-Ala ligand yields a value for the binding constant.     

Rational design of affinity peptide ligand by flexible docking simulation

Rational design of affinity peptide ligands of proteins by flexible docking simulation is performed using the SYBYL program package. This approach involves the use of experimental data to verify a scoring function that can be used to assess the affinity of a peptide for its target protein. The enzyme-linked immunosorbent assay (ELISA) data of several peptides displayed on phage surfaces for insulin and lysozyme, respectively, reported in literature are used for the purpose. It is found that the absolute values of the Dscore calculated from the docking correspond well to the ELISA data that relate to the affinity between the peptides and the target molecule. So, the Dscore function is used to assess the affinity of docked peptides in a pentapeptide library designed on the basis of protein (alpha-amylase) structure. As a result, a pentapeptide with a high Dscore value is selected and a hexapeptide (FHENWS) is built by linking serine to its C-terminal to lengthen the peptide. Molecular surface analysis with the MOLCAD program reveals that electrostatic interactions (including hydrogen bonds) and Van der Waals forces contribute to the affinity of the hexapeptide for alpha-amylase. Chromatographic experiments with the immobilized peptide have given further evidence for this observation. Adsorption isotherm described by the Langmuir equation indicates that the apparent binding constant of alpha-amylase to the immobilized hexapeptide was 2.5x10(5)L/mol. Finally, high affinity and specificity of the affinity adsorbent is exemplified by the purification of alpha-amylase from crude fermentation broth of Bacillus subtilis.