Essential of Proline and Valine Residues in the Peptide Derived from Lactoferrin for Angiotensin Converting Enzyme Inhibition

We synthesized Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu, the peptide contained in lactoferrin (Lf), to identify the angiotensin converting enzyme (ACE) inhibition. In an attempt to know the structure‐activity relationship of this peptide, we replaced Pro (the third amino acid residues from N‐terminal) or Val (the fourth amino acid residues from N‐terminal) with Ala (neutral amino acid), Glu (acidic amino acid) or Lys (basic amino acid) to produce six peptides. From the in vitro ACE inhibition (IC₅₀) of these synthesized peptides, the original peptide (Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu) showed higher ACE inhibition than the replaced six peptides. Thus, replacement of Pro at the third amino acid residues or Val at the fourth position with Ala, Glu or Lys revealed the ACE inhibition to be lower than the original form of Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu. Otherwise, we added one peptide at the C‐terminal of Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu and found both products with an addition of Val (Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu‐Val) or Ile (Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu‐Ile) showing a lower ACE inhibition than the original one. The ACE inhibitions produced by both replaced peptides were without significance. Also, deletion of the last peptide at the C‐terminal (Leu‐Arg‐Pro‐Val‐Ala‐Ala) failed to produce a marked change of ACE inhibition as compared to the original one. These results suggest that Pro and Val are essential in the peptide for inhibition of ACE activity.     

Affinity Maturation of Macrocyclic Peptide Modulators of Lys48-Linked Diubiquitin by a Twofold Strategy

Messenger RNA display of peptides containing non-proteinogenic amino acids, referred to as RaPID system, has become one of the leading methods to express libraries consisting of more than trillion-members of macrocyclic peptides, which allows for discovering de novo bioactive ligands. Ideal macrocyclic peptides should have dissociation constants (K_D) as low as single-digit values in the nanomolar range towards a specific target of interest. Here, a twofold strategy to discover optimized macrocyclic peptides within this affinity regime is described. First, benzyl thioether cyclized peptide libraries were explored to identify tight binding hits. To obtain more insights into critical sequence information, sequence alignment was applied to guide rational mutagenesis for the improvement of their binding affinity. Using this twofold strategy, benzyl thioether macrocyclic peptide binders against Lys48-linked ubiquitin dimer (K48-Ub2) were successfully obtained that display K_D values in the range 0.3–1.2 nm , which indicate binding two orders of magnitude stronger than those of macrocyclic peptides recently reported. Most importantly, this macrocyclic peptide also showed an improved cellular inhibition of the K48-Ub2 recognition by deubiquitinating enzymes and the 26S proteasome, resulting in the promotion of apoptosis in cancer cells.     

A general strategy to determine a target DNA sequence of a short peptide: application to a d-peptide.

Short peptides could potentially provide a novel element to read-out DNA sequences from the major groove. However, it is difficult to determine sequence-preference of de novo designed monomeric short peptides. Because DNS-binding affinity and specificity of short peptides are usually much lower than those of native DNA-binding proteins, determining the sequence-preference of short peptides by conventional methods utilized to deduce the target sequence of proteins often produces an unclear outcome. We report here a general strategy to defining the sequence-preference of a DNA-binding short peptide by using the heterodimers. A GCN4 basic region peptide tethers a low-affinity DNA-binding peptide adjacent to a GCN4 binding sequence through the cyclodextrin-adamantane association, thereby increasing local concentration of the low-affinity peptide on degenerated DNA sequences. An increase of the local concentration allows one to select a preferential sequence for the low-affinity DNA binding peptide. The method successfully identified specific sequences of short peptides derived from native DNA-binding proteins. The usefulness of this approach has been demonstrated by identifying preferred DNA targets for a peptide composed only of d-amino acids. The method is potentially applicable not only to artificial peptides, but also to other synthethic ligands.     

A pulsed field gradient NMR diffusion investigation of enkephalin peptide-sodium dodecyl sulfate micelle association

Pulsed field gradient NMR (PFG-NMR) diffusion experiments were used to investigate the binding of leucine and methionine enkephalin peptides to anionic sodium dodecyl sulfate (SDS) micelles. The study was undertaken to investigate the mechanism of interaction between enkephalin peptides and SDS micelles and to determine if NMR-derived association constants, K(eq), can predict the elution order in electrokinetic chromatography (EKC). In EKC, peptides are separated on the basis of their interactions with micelles. The Leu-enkephalin peptide-micelle association constant increased from 130 +/- 8 to 1459 +/- 57 and 1744 +/- 64 M(-1), respectively, when an Arg or Lys was added to the C-terminus. The association constant of Leu-enkephalinamide was approximately equal to that of Leu-enkephalin-Arg. Substitution of Phe4 with a Trp or Gly2 with an Ala in the Leu-enkephalin peptides also increased the micelle binding affinity. These results confirm that the interaction of Leu-enkephalin peptides with SDS micelles is largely electrostatic and that the non-polar amino acid side chains interact with the hydrophobic micelle core. The peptide-micelle association constants for the cationic Met-enkephalin peptides were also greater than their zwitterionic counterparts. For example, the Met-enkephalin K(eq) value was 162 +/- 9 M(-1), while the association constants for Met-enkephalin-Arg, Met-enkephalin-Lys, and Met-enkephalinamide were, respectively, 674 +/- 31, 426 +/- 23, and 453 +/- 27 M(-1). In both Met-enkephalin and Met-enkephalinamide, replacing Gly2 with an Ala did not significantly increase the association constant. These results confirm that with the Met-enkephalin peptides, there was little or no interaction of the amino acid side chains with the micelle core. For both the Leu-enkephalin and Met-enkephalin peptides, the association constants were consistent with EKC results, in that the peptides with smaller K(eq) values were found to elute before those with larger association constants.     

Simultaneous prediction of binding free energy and specificity for PDZ domain–peptide interactions

Interactions between protein domains and linear peptides underlie many biological processes. Among these interactions, the recognition of C-terminal peptides by PDZ domains is one of the most ubiquitous. In this work, we present a mathematical model for PDZ domain–peptide interactions capable of predicting both affinity and specificity of binding based on X-ray crystal structures and comparative modeling with Rosetta. We developed our mathematical model using a large phage display dataset describing binding specificity for a wild type PDZ domain and 91 single mutants, as well as binding affinity data for a wild type PDZ domain binding to 28 different peptides. Structural refinement was carried out through several Rosetta protocols, the most accurate of which included flexible peptide docking and several iterations of side chain repacking and backbone minimization. Our findings emphasize the importance of backbone flexibility and the energetic contributions of side chain-side chain hydrogen bonds in accurately predicting interactions. We also determined that predicting PDZ domain–peptide interactions became increasingly challenging as the length of the peptide increased in the N-terminal direction. In the training dataset, predicted binding energies correlated with those derived through calorimetry and specificity switches introduced through single mutations at interface positions were recapitulated. In independent tests, our best performing protocol was capable of predicting dissociation constants well within one order of magnitude of the experimental values and specificity profiles at the level of accuracy of previous studies. To our knowledge, this approach represents the first integrated protocol for predicting both affinity and specificity for PDZ domain–peptide interactions.     

Design and synthesis of peptides that bind alpha-bungarotoxin with high affinity

BACKGROUND: Alpha-bungarotoxin (alpha-BTX) is a highly toxic snake venom alpha-neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. We describe the design and synthesis of peptides that bind alpha-BTX with high affinity, and inhibit its interaction with AChR with an IC(50) of 2 nM. The design of these peptides was based on a lead peptide with an IC(50) of 3x10(-7) M, previously identified by us [M. Balass et al., Proc. Natl. Acad. Sci. USA 94 (1997) 6054] using a phage-display peptide library. RESULTS: Employing nuclear magnetic resonance-derived structural information [T. Scherf et al., Proc. Natl. Acad. Sci. USA 94 (1997) 6059] of the complex of alpha-BTX with the lead peptide, as well as structure-function analysis of the ligand-binding site of AChR, a systematic residue replacement of the lead peptide, one position at a time, yielded 45 different 13-mer peptides. Of these, two peptides exhibited a one order of magnitude increase in inhibitory potency in comparison to the lead peptide. The design of additional peptides, with two or three replacements, resulted in peptides that exhibited a further increase in inhibitory potency (IC(50) values of 2 nM), that is more than two orders of magnitude better than that of the original lead peptide, and better than that of any known peptide derived from AChR sequence. The high affinity peptides had a protective effect on mice against alpha-BTX lethality. CONCLUSIONS: Synthetic peptides with high affinity to alpha-BTX may be used as potential lead compounds for developing effective antidotes against alpha-BTX poisoning. Moreover, the procedure employed in this study may serve as a general approach for the design and synthesis of peptides that interact with high affinity with any desired biological target.     

Model peptides based on the binding loop of the copper metallochaperone Atx1: selectivity of the consensus sequence MxCxxC for metal ions Hg(II), Cu(I), Cd(II), Pb(II), and Zn(II)

The amino acid sequence MxCxxC is conserved in many soft-metal transporters that are involved in the control of the intracellular concentration of ions such as Cu(I), Hg(II), Zn(II), Cd(II), and Pb(II). A relevant task is thus the selectivity of the motif MxCxxC for these different metal ions. To analyze the coordination properties and the selectivity of this consensus sequence, we have designed two model peptides that mimic the binding loop of the copper chaperone Atx1: the cyclic peptide P(C) c(GMTCSGCSRP) and its linear analogue P(L) (Ac-MTCSGCSRPG-NH2). By using complementary analytical and spectroscopic methods, we have demonstrated that 1:1 complexes are obtained with Cu(I) and Hg(II), whereas 1:1 and 1:2 (M:P) species are successively formed with Zn(II), Cd(II), and Pb(II). The complexation properties of the cyclic and linear peptides are very close, but the cyclic compound provides systematically higher affinity constants than its unstructured analogue. The introduction of a xPGx motif that forms a type II beta turn in P(C) induces a preorganization of the binding loop of the peptide that enhances the stabilities of the complexes (up to 2 orders of magnitude difference for the Hg complexes). The affinity constants were measured in the absence of any reducing agent that would compete with the peptides and range in the order Hg(II) > Cu(I) > Cd(II) > Pb(II) > Zn(II). This sequence is thus highly selective for Cu(I) compared to the essential ion Zn(II) that could compete in vivo or compared to the toxic ions Cd(II) and Pb(II). Only Hg(II) may be an efficient competitor of Cu(I) for binding to the MxCxxC motif in metalloproteins.     

Organic Ligand Binding by a Hydrophobic Cavity in a Designed Tetrameric Coiled‐Coil Protein

The design and characterization of a hydrophobic cavity in de novo designed proteins provides a wide range of information about the functions of de novo proteins. We designed a de novo tetrameric coiled‐coil protein with a hydrophobic pocketlike cavity. Tetrameric coiled coils with hydrophobic cavities have previously been reported. By replacing one Leu residue at the a position with Ala, hydrophobic cavities that did not flatten out due to loose peptide chains were reliably created. To perform a detailed examination of the ligand‐binding characteristics of the cavities, we originally designed two other coiled‐coil proteins: AM2, with eight Ala substitutions at the adjacent a and d positions at the center of a bundled structure, and AM2W, with one Trp and seven Ala substitutions at the same positions. To increase the association of the helical peptides, each helical peptide was connected with flexible linkers, which resulted in a single peptide chain. These proteins exhibited CD spectra corresponding to superhelical structures, despite weakened hydrophobic packing. AM2W exhibited binding affinity for size‐complementary organic compounds. The dissociation constants, K_d, of AM2W were 220 nM for adamantane, 81 μM for 1‐adamantanol, and 294 μM for 1‐adamantaneacetic acid, as measured by fluorescence titration analyses. Although it was contrary to expectations, AM2 did not exhibit any binding affinity, probably due to structural defects around the designed hydrophobic cavity. Interestingly, AM2W exhibited incremental structure stability through ligand binding. Plugging of structural defects with organic ligands would be expected to facilitate protein folding.     

Arginine- and lysine-specific polymers for protein recognition and immobilization

Free radical polymerization of methacrylamide-based bisphosphonates turns weak arginine binders into powerful polymeric protein receptors. Dansyl-labeled homo- and copolymers with excellent water solubility are accessible through a simple copolymerization protocol. Modeling studies point to a striking structural difference between the stiff rodlike densely packed homopolymer 1 and the flexible copolymer 2 with spatially separated bisphosphonate units. Fluorescence titrations in buffered aqueous solution (pH = 7.0) confirm the superior affinity of the homopolymer toward oligoarginine peptides reaching nanomolar K(D) values for the Tat peptide. Basic proteins are bound almost equally well by 1 and 2 with micromolar affinities, with the latter producing much more soluble complexes. The Arg selectivity of the monomer is transferred to the polymer, which binds Arg-rich proteins 1 order of magnitude tighter than lysine-rich pendants of comparable pI, size, and (Arg/Lys vs Glu/Asp) ratio. Noncovalent deposition of both polymers on glass substrates via polyethyleneimine layers results in new materials suitable for peptide and protein immobilization. RIfS measurements allow calculation of association constants K(a) as well as dissociation kinetics k(D). They generally confirm the trends already found in free solution. Close inspection of electrostatic potential surfaces suggest that basic domains favor protein binding on the flat surface. The high specificity of the bisphosphonate polymers toward basic proteins is demonstrated by comparison with polyvinyl sulfate, which has almost no effect in RIfS experiments. Thus, copolymerization of few different comonomer units without cross-linking enables surface recognition of basic proteins in free solution as well as their effective immobilization on surfaces.     

A peptide motif recognizing a polymer stereoregularity

A combinatorial phage display method was applied to films composed of a stereoregular polymer of methacrylates. The phage clones with selective affinity for isotactic (it) poly(methyl methacrylate) (PMMA) were isolated. Greater amounts of the phage clones bound to it-PMMA, compared to other stereoregular PMMAs. The phage expressing ELWRPTR most strongly bound to the polymer, and the selectivity was also the best. The peptide motif essential for the specific interaction with the stereoregular polymer was revealed.     

Peptide epitope-imprinted polymer microarrays for selective protein recognition. Application for SARS-CoV-2 RBD protein

We introduce a practically generic approach for the generation of epitope-imprinted polymer-based microarrays for protein recognition on surface plasmon resonance imaging (SPRi) chips. The SPRi platform allows the subsequent rapid screening of target binding kinetics in a multiplexed and label-free manner. The versatility of such microarrays, both as synthetic and screening platform, is demonstrated through developing highly affine molecularly imprinted polymers (MIPs) for the recognition of the receptor binding domain (RBD) of SARS-CoV-2 spike protein. A characteristic nonapeptide GFNCYFPLQ from the RBD and other control peptides were microspotted onto gold SPRi chips followed by the electrosynthesis of a polyscopoletin nanofilm to generate in one step MIP arrays. A single chip screening of essential synthesis parameters, including the surface density of the template peptide and its sequence led to MIPs with dissociation constants (K_D) in the lower nanomolar range for RBD, which exceeds the affinity of RBD for its natural target, angiotensin-convertase 2 enzyme. Remarkably, the same MIPs bound SARS-CoV-2 virus like particles with even higher affinity along with excellent discrimination of influenza A (H3N2) virus. While MIPs prepared with a truncated heptapeptide template GFNCYFP showed only a slightly decreased affinity for RBD, a single mismatch in the amino acid sequence of the template, i.e. the substitution of the central cysteine with a serine, fully suppressed the RBD binding.

We introduce highly affine epitope-imprinted polymer-based microarrays for selective protein detection by surface plasmon resonance imaging as shown through the selective recognition of the receptor binding domain of SARS-CoV-2 spike protein.     

Molecular dynamics simulations of pro-apoptotic BH3 peptide helices in aqueous medium: relationship between helix stability and their binding affinities to the anti-apoptotic protein Bcl-X<Subscript>L</Subscript>

The B-cell lymphoma 2 (Bcl-2) family of proteins regulates the intrinsic pathway of apoptosis. Interactions between specific anti- and pro-apoptotic Bcl-2 proteins determine the fate of a cell. Anti-apoptotic Bcl-2 proteins have been shown to be over-expressed in certain cancers and they are attractive targets for developing anti-cancer drugs. Peptides from the BH3 region of pro-apoptotic proteins have been shown to interact with anti-apoptotic Bcl-2 proteins and induce biological activity similar to that observed in parent proteins. However, the specificity of BH3 peptides derived from different pro-apoptotic proteins differ for different anti-apoptotic Bcl-2 proteins. In this study, we have investigated the relationship between the stable helical nature of BH3 peptides and their affinities to Bcl-X_L, an anti-apoptotic Bcl-2 protein. We have carried out molecular dynamics simulations of six BH3 peptides derived from Bak, Bad and Bim pro-apoptotic proteins for a period of 50 ns each in aqueous medium. Due to the amphipathic nature of BH3 peptides, the hydrophobic residues on the hydrophobic face tend to cluster together in all BH3 peptides. While this process resulted in a complete loss of helical structure in 16-mer Bak and 16-mer Bad wild type peptides, stabilizing interactions in the hydrophilic face of the BH3 peptides and capping interactions helped to maintain partial helical character in 16-mer Bad mutant and 16-mer Bim peptides. The latter two 16-mer peptides exhibit higher affinity for Bcl-X_L. Similarly the longer BH3 peptides, 25-mer Bad and 33-mer Bim, also resulted in smaller and stable helical fragments and their helical conformation is stabilized by interactions between residues in the solvent-exposed hydrophilic half of the peptide. The stable nature of helical segment in a BH3 peptide can be directly correlated to its binding affinity and the helical region encompassed the highly conserved Leu residue. We propose that upon approaching the hydrophobic groove of anti-apoptotic proteins, a longer helix will be induced in high affinity BH3 peptides by extending the smaller stable helical segments around the conserved Leu residue in both N- and C-terminal regions. The results reported in this study will have implications in developing peptide-based inhibitors for anti-apoptotic Bcl-2 proteins.     

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.     

Novel strategy for the design of a new zinc finger: creation of a zinc finger for the AT-rich sequence by alpha-helix substitution

In this communication, a novel strategy for the design of a zinc finger peptide on the basis of alpha-helix substitution has been demonstrated. Sp1HM is a helix-substituted mutant for the wild-type Sp1(zf123) and its alpha-helix of each finger is replaced by that of fingers 4-6 of CF2-II. The circular dichroism spectrum of Sp1HM suggests that Sp1HM has an ordered secondary structure similar to that of Sp1(zf123). From the analyses of the DNA binding affinity and specificity by gel mobility shift assay, it is clearly indicated that Sp1HM specifically binds to the AT-rich sequence (5'-GTA TAT ATA-3') with 3.2 nM dissociation constants. Moreover, the zinc finger peptides for the sequence alternating between the AT- and GC-rich subsites can also be created by the alpha-helix substitution. This strategy is evidently effective and is also more convenient than the phage display method. Consequently, our design method is widely applicable to creating zinc finger peptides with novel binding specificities.     

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.     

Energetic switch of the proline effect in collision‐induced dissociation of singly and doubly protonated peptide Ala‐Ala‐Arg‐Pro‐Ala‐Ala

Suppression of the selective cleavage at N‐terminal of proline is observed in the peptide cleavage by proteolytic enzyme trypsin and in the fragment ion mass spectra of peptides containing Arg‐Pro sequence. An insight into the fragmentation mechanism of the influence of arginine residue on the proline effect can help in prediction of mass spectra and in protein structure analysis. In this work, collision‐induced dissociation spectra of singly and doubly charged peptide AARPAA were studied by ESI MS/MS and theoretical calculation methods. The proline effect was evaluated by comparing the experimental ratio of fragments originated from cleavage of different amide bonds. The results revealed that the backbone amide bond cleavage was selected by the energy barrier height of the fragmentation pathway although the strong proton affinity of the Arg side chain affected the stereostructure of the peptide and the dissociation mechanism. The thermodynamic stability of the fragment ions played a secondary role in the abundance ratio of fragments generated via different pathways. Fragmentation studies of protonated peptide AACitPAA supported the energy‐dependent hypothesis. The results provide an explanation to the long‐term arguments between the steric conflict and the proton mobility mechanisms of proline effect.     

Selection of New Sequence‐Selective Unnatural Peptides Binding to Double‐Stranded Deoxyribonucleic Acids (dsDNA) by Means of a Gel‐Retardation Experiment for Library Analysis

Previously, we developed a methodology for the solid‐phase screening of peptide libraries for interaction with double‐stranded deoxyribonucleic acids (dsDNA). In the search for new and more‐potent DNA ligands, we investigated the strategy of solution‐phase screening of chemical libraries consisting of unnatural oligopeptides. After synthesis of the selected amino acid building blocks, libraries were constructed with the general structure Ac‐Arg‐Ual‐Sar‐X¹‐X²‐X³‐Arg‐NH₂, where X represents each of twelve unnatural or natural amino acids. Optimization of the sequence of binding peptides was performed with an iterative deconvolution procedure. Selection of interacting peptides was carried out in solution by means of gel‐retardation experiments, starting with libraries of 144 compounds. A 14‐base‐pair double‐stranded DNA fragment was chosen as the target. After several cycles of synthesis and screening of libraries and individual peptides, an oligopeptide was selected with an apparent dissociation constant of 9⋅10⁻⁵ M , as determined by gel‐retardation experiments. This peptide was studied by NMR spectroscopy. A certain degree of conformational pre‐organization of the peptides was shown by temperature‐dependent circular‐dichroism experiments. Finally, DNase‐I‐footprinting studies indicated a preferential interaction with a 6‐base‐pair mixed sequence 5′‐CTGCAT‐3′. This study demonstrates that gel‐shift experiments can be used for the solution‐phase screening of library mixtures of peptides against dsDNA. In general, this technique allows the selection of new sequence‐selective dsDNA‐interacting molecules. Furthermore, novel dsDNA‐binding unnatural oligopeptides were developed with affinities in the 0.1 mM range.     

Investigation of the interactions between ginsenosides and amino acids by mass spectrometry and theoretical chemistry

In order to evaluate the essence of the interactions of ginsenosides and proteins which are composed by α-amino acids, electrospray ionization mass spectrometry was employed to study the noncovalent interactions between ginsenosides (Rb₂, Rb₃, Re, Rg₁ and Rh₁) and 18 kinds of α-amino acids (Asp, Glu, Asn, Phe, Gln, Thr, Ser, Met, Trp, Val, Gly, Ile, Ala, Leu, Pro, His, Lys and Arg). The 1:1 and 2:1 noncovalent complexes of ginsenosides and amino acids were observed in the mass spectra. The dissociation constants for the noncovalent complexes were directly calculated based on peak intensities of ginsenosides and the noncovalent complexes in the mass spectra. Based on the dissociation constants, it can be concluded that the acidic and the basic amino acids, Asp, Glu, Lys and Arg, bound to ginsenosides more strongly than other amino acids. The experimental results were verified by theoretical calculations of parameters of noncovalent interaction between ginsenoside Re and Arg which served as a representative example. Two kinds of binding forms, “head–tail” (“H–T”) and “head–head” (“H–H”), were proposed to explain the interaction between ginsenosides and amino acids. And the interaction in “H–T” form was stronger than that in “H–H” form.     

从组合化学肽库中筛选亲和配基

在亲和色谱研究中 ,建立适合于某种分离对象的亲和色谱体系的关键是寻找适合的亲和配基 .小肽亲和配基具有性质稳定、合成简单、价格低及生物相容性好等特点[1] ,但这类配基也存在着亲和力弱或选择性低的缺点 .因此 ,如何寻找小肽配基以及如何提高小肽配基的亲和力和选择性的问题引起了人们的关注与重视 [1～ 3 ] .组合化学法是一种快速制备大量相关或同类化合物的革新性的方法 [4 ] ,组合肽库包括噬菌体展示肽库和合成肽库 ,这两类肽库均可用于小肽配基的筛选与优化 [1,5] .组合化学法尽管有效 ,但以小肽为目的物进行筛选时 ,往往因肽 -肽…     

Negative Ion In-Source Decay Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Sequencing Acidic Peptides

Matrix-assisted laser desorption/ionization (MALDI) in-source decay was studied in the negative ion mode on deprotonated peptides to determine its usefulness for obtaining extensive sequence information for acidic peptides. Eight biological acidic peptides, ranging in size from 11 to 33 residues, were studied by negative ion mode ISD (nISD). The matrices 2,5-dihydroxybenzoic acid, 2-aminobenzoic acid, 2-aminobenzamide, 1,5-diaminonaphthalene, 5-amino-1-naphthol, 3-aminoquinoline, and 9-aminoacridine were used with each peptide. Optimal fragmentation was produced with 1,5-diaminonphthalene (DAN), and extensive sequence informative fragmentation was observed for every peptide except hirudin(54–65). Cleavage at the N–Cα bond of the peptide backbone, producing c′ and z′ ions, was dominant for all peptides. Cleavage of the N–Cα bond N-terminal to proline residues was not observed. The formation of c and z ions is also found in electron transfer dissociation (ETD), electron capture dissociation (ECD), and positive ion mode ISD, which are considered to be radical-driven techniques. Oxidized insulin chain A, which has four highly acidic oxidized cysteine residues, had less extensive fragmentation. This peptide also exhibited the only charged localized fragmentation, with more pronounced product ion formation adjacent to the highly acidic residues. In addition, spectra were obtained by positive ion mode ISD for each protonated peptide; more sequence informative fragmentation was observed via nISD for all peptides. Three of the peptides studied had no product ion formation in ISD, but extensive sequence informative fragmentation was found in their nISD spectra. The results of this study indicate that nISD can be used to readily obtain sequence information for acidic peptides.

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