Minimal Binding Sequence Research Articles

Isolation of Peptide Ligands for the HIV Capsid Protein p24 by Phage-Display

PurposeIsolate renewable and cost-efficient affinity reagents that will facilitate the detection of p24, the capsid protein of Human Immunodeficiency Virus (HIV), by screening phage-displayed combinatorial peptide libraries and identifying peptide ligands.MethodFour in-house combinatorial peptide libraries were screened for binders in three progressive rounds against monomeric p24 protein. Peptide binders were characterized by ELISA and Surface Plasmon Resonance (SPR) and one peptide sequence was evaluated in a lateral flow assay (LFA).ResultWe identified 26 unique peptide sequences that exhibit varying phage ELISA signals above background for p24. We subsequently validated the binding of one linear and two cyclized peptide sequences with synthetic peptides. Alanine-scanning identified several residues critical to binding in the linear peptide. The linear peptide could be used for p24 detection in ELISA and LFAs.ConclusionPhage-displayed combinatorial peptide libraries are suitable for isolation of binders against p24 and potentially other targets. Upon identification of a minimal binding sequence, the subsequent characterization and future optimization of it can lead to a variety of diagnostic assays.

Portability of a Small-Molecule Binding Site between Disordered Proteins.

Intrinsically disordered proteins (IDPs) are important in both normal and disease states. Small molecules can be targeted to disordered regions, but we currently have only a limited understanding of the nature of small-molecule binding sites in IDPs. Here, we show that a minimal small-molecule binding sequence of eight contiguous residues derived from the Myc protein can be ported into a different disordered protein and recapitulate small-molecule binding activity in the new context. We also find that the residue immediately flanking the binding site can have opposing effects on small-molecule binding in the different disordered protein contexts. The results demonstrate that small-molecule binding sites can act modularly and are portable between disordered protein contexts but that residues outside of the minimal binding site can modulate binding affinity.

Multianalytical Study of the Binding between a Small Chiral Molecule and a DNA Aptamer: Evidence for Asymmetric Steric Effect upon 3'- versus 5'-End Sequence Modification.

Nucleic acid aptamers are involved in a broad field of applications ranging from therapeutics to analytics. Deciphering the binding mechanisms between aptamers and small ligands is therefore crucial to improve and optimize existing applications and to develop new ones. Particularly interesting is the enantiospecific binding mechanism involving small molecules with nonprestructured aptamers. One archetypal example is the chiral binding between l-tyrosinamide and its 49-mer aptamer for which neither structural nor mechanistic information is available. In the present work, we have taken advantage of a multiple analytical characterization strategy (i.e., using electroanalytical techniques such as kinetic rotating droplet electrochemistry, fluorescence polarization, isothermal titration calorimetry, and quartz crystal microbalance) for interpreting the nature of binding process. Screening of the binding thermodynamics and kinetics with a wide range of aptamer sequences revealed the lack of symmetry between the two ends of the 23-mer minimal binding sequence, showing an unprecedented influence of the 5' aptamer modification on the bimolecular binding rate constant kon and no significant effect on the dissociation rate constant koff. The results we have obtained lead us to conclude that the enantiospecific binding reaction occurs through an induced-fit mechanism, wherein the ligand promotes a primary nucleation binding step near the 5'-end of the aptamer followed by a directional folding of the aptamer around its target from 5'-end to 3'-end. Functionalization of the 5'-end position by a chemical label, a polydA tail, a protein, or a surface influences the kinetic/thermodynamic constants up to 2 orders of magnitude in the extreme case of a surface immobilized aptamer, while significantly weaker effect is observed for a 3'-end modification. The reason is that steric hindrance must be overcome to nucleate the binding complex in the presence of a modification near the nucleation site.

Polymerisation of a T Cell Epitope with an Immunostimulatory C3d Peptide Sequence Enhances Antigen Specific T Cell Responses

The complement protein C3d and C3d derived peptides that bind CD21 are known to enhance immunity to co-immunised antigens. In this study we have synthesised the minimal CD21 binding sequence of C3d (1227LYNVEA1232) as mono, di and tri tandem repeats and derivatised the N-terminus with an acryloyl moiety. These acryloyl-(C3d) n peptides were co-polymerised with a acryloyl-T cell epitope (PAS1K) from the Porphyromonas gingivalis antigen the RgpA–Kgp proteinase–adhesin complex. The ability of C3d containing polymers to enhance T cell immunity in vitro and in vivo was evaluated. When used to stimulate in vitro PAS1K-primed or RgpA–Kgp complex-primed T cells the C3d containing PAS1K polymers induced a mixed and significantly (p < 0.05) higher IL-4 and IFNγ T cell response compared to that induced by the PAS1K peptide or polymer. PAS1K polymers containing tandem repeats of C3d induced a significantly (p < 0.05) stronger maximal proliferative response, at the same antigenic dose, compared to that induced by the PAS1K peptide or polymer. When used as immunogens to prime T cells all of the C3d containing PAS1K polymers induced a dominant IFNγ T cell response and reduced the antigen dose required for maximal proliferation 150-fold compared to that required for the PAS1K-peptide or polymer primed T cells. In conclusion, the 6 residue sequence LYNVEA from C3d is sufficient to enhance immunity to an antigen and that the effect is more pronounced when C3d is part of the immunising antigen rather than an in vitro stimulating antigen.

Opioid Activity Profiles of Oversimplified Peptides Lacking in the Protonable N-Terminus

Recently, we described cyclopeptide opioid agonists containing the d-Trp-Phe sequence. To expand the scope of this atypical pharmacophore, we tested the activity profiles of the linear peptides Ac-Xaa-Phe-Yaa (Xaa = l/d-Trp, d-His/Lys/Arg; Yaa = H, GlyNH(2)). Ac-d-Trp-PheNH(2) appeared to be the minimal binding sequence, while Ac-d-Trp-Phe-GlyNH(2) emerged as the first noncationizable short peptide (partial) agonist with high μ-opioid receptor affinity and selectivity. Conformational analysis suggested that 5 adopts in solution a β-turn conformation.

Insights into the Dynamics and Actin Interaction of the Intrinsically Disordered Tarp Protein from Chlamydia

The human pathogen Chlamydia trachomatis, and all species within the Chlamydiaceae bacterial family, are prokaryotic obligate intracellular parasites. Their host cell internalisation mechanism is well conserved and includes a type-III secreted effector termed Tarp (Translocated actin recruiting protein). Once within the host cell, Tarp is able to nucleate actin filaments and induce a phagocytosis-like Chlamydial encapsulation. While most of Tarp is predicted to be intrinsically disordered, the minimal actin binding sequence from Tarp (100 residues long) contains a short helix which shows homology with the actin binding WH2 motif from WAVE2, maintaining 9 conserved residues across a 15-residue stretch. Using nuclear magnetic resonance (NMR) spectroscopy and the minimal actin binding region, we have determined the residues affected by the Tarp:actin interaction. X-ray structures of other WH2:actin complexes show a short helix binding within the actin hydrophobic cleft and a region of 15-20 extended residues lying along the actin surface. Despite limited sequence homology it is therefore possible that Tarp is binding actin with similar helical and extended elements. Based upon the affected residues of the 100 residue fragment, an 83 residue mutant has been synthesised which still contains the residues affected upon the Tarp:actin interaction. Data from NMR studies of this minimised Tarp fragment shows it is also able to bind G-actin in vitro. Furthermore, NMR spectra of both Tarp fragments alone indicates that in solution they exists as disordered polypeptides. T1 and heteronuclear {1H-15N} NOE relaxation studies of the fragments suggest that a region of decreased dynamics exists which corresponds to the WH2-like helix.

Mutually Exclusive Cytoplasmic Dynein Regulation by NudE-Lis1 and Dynactin

Cytoplasmic dynein is responsible for a wide range of cellular roles. How this single motor protein performs so many functions has remained a major outstanding question for many years. Part of the answer is thought to lie in the diversity of dynein regulators, but how the effects of these factors are coordinated in vivo remains unexplored. We previously found NudE to bind dynein through its light chain 8 (LC8) and intermediate chain (IC) subunits (1), the latter of which also mediates the dynein-dynactin interaction (2). We report here that NudE and dynactin bind to a common region within the IC, and compete for this site. We find LC8 to bind to a novel sequence within NudE, without detectably affecting the dynein-NudE interaction. We further find that commonly used dynein inhibitory reagents have broad effects on the interaction of dynein with its regulatory factors. Together these results reveal an unanticipated mechanism for preventing dual regulation of individual dynein molecules, and identify the IC as a nexus for regulatory interactions within the dynein complex.

Using Self-Assembled Monolayers To Understand α8β1-Mediated Cell Adhesion to RGD and FEI Motifs in Nephronectin

Nephronectin is an extracellular matrix protein that interacts with the α8β1 integrin receptor and plays a role in tissue and organ development, though the motifs that mediate adhesion to the receptor remain unclear. This paper describes the use of self-assembled monolayers to study the adhesion of α8β1-presenting cells to the RGD and DLFEIFEIER ligands in nephronectin and found that both ligands can independently mediate cell adhesion through nonoverlapping binding sites on the integrin. Peptide truncation experiments showed FEI to be the minimal binding sequence within the DLFEIFEIER sequence, and adhesion experiments with peptides that include both the RGD and FEI sequences demonstrate that the two peptides bind synergistically to the receptor. Finally, a peptide array was used to establish a strict requirement for the glutamate residue of FEI and tolerance of other aromatic and hydrophobic residues in the first and third positions, respectively. This work provides an enhanced understanding of the binding of nephronectin with α8β1 and identifies a peptide ligand that can be used for targeting the α8β1 integrin.

DNA‐binding properties and telomere function of hnRNP A3

Human telomeric DNA consists of hundreds to thousands of tandem repeat hexanucleotides (TTAGGG) and is bound by telomere‐associated proteins to form specialized structure that maintains the integrity of chromosome. These telomere‐associated proteins serve as structural and/or regulatory components for telomere functions. Dysfunctional telomeres are known to trigger growth arrest, senescence, and apoptosis. Previously, we have identified hnRNP A3, a poorly understood member of the hnRNPs A/B, as a novel telomere‐associated protein that binds specifically to single‐stranded telomeric DNA. In this study, the DNA‐binding properties of hnRNP A3 in vitro as well as its putative role of telomere maintenance in vivo were investigated. Here, we showed that the minimal binding sequence (MBS) for hnRNP A3 is 5′‐TAGGGTTAGGG‐3′, and the position of MBS in the single‐strand DNA had little or no effect on the binding by hnRNP A3. The consensus binding sequence in the 11‐mer MBS was characterized as 5′‐[T/C]AG[G/T]NN[T/C]AG[G/T]N‐3′. Furthermore, confocal microscopy and chromatin‐immunoprecipitation (ChIP) studies revealed that hnRNP A3 are associated with telomere in vivo. Knocking‐ down the expression of hnRNP A3 had no effect on telomere length maintenance and did not affect cell proliferation capacity. However, overexpression of hnRNPA3 resulted in the production of steady‐state short telomeres in several cell lines. These findings indicate that hnRNP A3 can participate in telomere function and acts as a negative regulator of telomere length maintenance. This work was supported by research grant CMRPD 170541.

The Platelet Integrin αIIbβ3 Binds to the RGD and AGD Motifs in Fibrinogen

Fibrinogen (Fbg) mediates platelet aggregation by binding the alphaIIbbeta3 integrin receptor, but the interaction of the receptor with peptide motifs of Fbg remains unresolved. This paper describes the use of self-assembled monolayers (SAMs) to study the adhesion of alphaIIbbeta3-transfected CHO cells to the GRGDS and HHLGGAKQAGDV motifs within Fbg. Cells adhered to and spread on monolayers presenting either peptide. Cell adhesion could be inhibited by either soluble peptide, demonstrating that the peptides bind competitively to the integrin. A peptide array was used to show that AGD was the minimal binding sequence in HHLGGAKQAGDV and that the receptor recognizes ligands of the form GXGDSC, where X is a hydrophobic or basic residue. This work revises our understanding of the alphaIIbbeta3 specificity and also suggests a new class of antithrombotic agents.

Changes in Conformational Dynamics of mRNA upon AtGRP7 Binding Studied by Fluorescence Correlation Spectroscopy

The clock-regulated RNA recognition motif (RRM)-containing protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein) influences the amplitude of its transcript oscillation at the post-transcriptional level. This autoregulation relies on AtGRP7 binding to its own pre-mRNA. The sequence and structural requirements for this interaction are unknown at present. In this work, we used photoinduced electron transfer fluorescence correlation spectroscopy (PET-FCS) as a novel technique to study the role of target RNA secondary structure and conformational dynamics during the recognition and binding process. Conformational dynamics of single-stranded (ss) oligonucleotides were studied in aqueous solution with single-molecule sensitivity and high temporal resolution by monitoring fluorescence quenching of the oxazine fluorophore MR121 by guanosine residues. Comparative analysis of translational diffusion constants revealed that both ssRNA and ssDNA bind to AtGRP7 with similar dissociation constants on the order of 10(-7) M and that a minimal binding sequence 5'-UUC UGG-3' is needed for recognition by AtGRP7. PET-FCS experiments demonstrated that conformational flexibility of short, single-stranded, MR121-labeled oligonucleotides is reduced upon AtGRP7 binding. In contrast to many other RRM proteins, AtGRP7 binds to ssRNA preferentially if the RNA is fully stretched and not embedded within a stable secondary structure. The results suggest that AtGRP7 binding leads to a conformational rearrangement in the mRNA, arresting the flexible target sequence in an extended structure of reduced flexibility that may have consequences for further post-transcriptional processing of the mRNA.

TraM protein of plasmid R1: In vitro selection of the target region reveals two consensus 7 bp binding motifs spaced by a 4 bp linker of defined sequence

The TraM protein encoded by plasmid R1 has three functions in conjugative transfer: (i) it positively controls transfer gene expression, (ii) it stimulates efficient site-specific ssDNA cleavage at the oriT in vivo and (iii) it couples the relaxosome to the envelope-bound transport complex. Plasmid R1 contains two binding regions for TraM, sbmA and sbmB, either of which comprises several minimal TraM targets. SELEX of a randomized minimal (18 bp) TraM binding sequence was used to search for sequences that bind TraM most efficiently. This approach resulted in a sequence with a modular structure with two 7 bp consensus motifs 5′ T 1G 2A 3N 4T 5C 6R 7 and 5′ T 1G 2A 3N 4T 5Y 6R 7 spaced by a 4 bp linker with the consensus sequence 5′ Y 1A 2/C 2T 3/G 3A 4. EMSAs revealed that evolution in vivo was for maximal binding affinity. R1 derivatives with mutated sbmA and sbmB sites based on one in vitro selected sequence were found to be either 5-fold impaired ( mut sbmA) in conjugation or could not be transferred by conjugation anymore ( mut sbmB). Transcriptional lacZ fusions demonstrated that the introduced sbmB mutations affected promoter activity severely and abrogated autoregulation by TraM, thereby explaining this drastic effect on conjugal transfer. The implications of these findings are discussed in the context of the role of TraM.

Epitope Mapping of PR81 anti-MUC1 Monoclonal Antibody Following PEPSCAN and Phage Display Techniques

PR81 is an anti-MUC1 monoclonal antibody (MAb) which was generated against human MUC1 mucin that reacted with breast cancerous tissue, MUC1 positive cell line (MCF-7, BT-20, and T-4 7 D), and synthetic peptide, including the tandem repeat sequence of MUC1. Here we characterized the binding properties of PR81 against the tandem repeat of MUC1 by two different epitope mapping techniques, namely, PEPSCAN and phage display. Epitope mapping of PR81 MAb by PEPSCAN revealed a minimal consensus binding sequence, PDTRP, which is found on MUC1 peptide as the most important epitope. Using the phage display peptide library, we identified the motif PD(T/S/G)RP as an epitope and the motif AVGLSPDGSRGV as a mimotope recognized by PR81. Results of these two methods showed that the two residues, arginine and aspartic acid, have important roles in antibody binding and threonine can be substituted by either glycine or serine. These results may be of importance in tailor making antigens used in immunoassay.

Identification of the von Willebrand Factor Binding Site in Collagen Using Triple Helical Peptides.

The interaction of the plasma protein von Willebrand factor (VWF) with subendothelial collagen initiates adhesion of blood platelets to the damaged vessel wall or ruptured atherosclerotic plaque. A detailed molecular description of the VWF-collagen interaction may facilitate development of a novel class of antithrombotic drugs that inhibits this vital step in platelet thrombus formation. We have previously used site-directed mutagenesis to map the collagen-binding site in the VWF A3 domain. Here, we report the identification of a 9-aminoacid sequence in collagen type III which mediates VWF binding. We have synthesized a set of 57 peptides, each containing 27 amino acids of native collagen sequence flanked at each end by five GPP (standard amino acid nomenclature) triplets which support the triple helical structure that is essential for ligand recognition by collagen. The sequence of each peptide overlaps by 9 amino acids with that of each adjacent peptide. A single peptide from this set (#23) was shown to bind VWF in a solid-phase binding assay. The affinity of peptide #23 for VWF was comparable to that of native collagen type III. The peptide #23-VWF interaction was abolished by a monoclonal antibody directed against the collagen-binding site on the VWF A3 domain. Furthermore, recombinant VWF variants that were previously shown to lack collagen-binding capacity (delta A3, His1023Ala) were not able to bind to the peptide. Using surface plasmon resonance, we showed that the peptide bound strongly to the isolated VWF A3 domain (Kd 300 nM). Immobilized peptide #23 also supported platelet adhesion from whole blood under flow conditions and washed platelet adhesion under static conditions. Platelet adhesion to peptide #23 could be abrogated by a monoclonal antibody directed against the VWF A3 domain, which inhibits the interaction of VWF with full-length collagen. We subsequently synthesized a set of truncated and alanine-modified triple helical peptides based on the sequence of #23, which were all tested for VWF and platelet binding from whole blood under flow conditions. Modified peptides either strongly interacted with both VWF and platelets, or lacked both VWF and platelet binding. Based on these experiments, we identified the sequence RGQOGVMGF (O is hydroxyproline) as the minimal VWF binding sequence in collagen type III. Mutation of either Q or M to alanine (A) did not affect VWF binding, whereas replacement of R, O, V, and F by A completely abolished VWF binding. Glycine residues were not replaced, as they are essential for triple helix formation. A model of the VWF A3 domain with this nonapeptide collagen sequence was constructed to give detailed insight into the VWF-collagen interaction. In conclusion, we have identified a 9 amino acid sequence in collagen type III that is entirely responsible for high affinity binding to VWF. The detailed molecular description of the VWF-collagen interaction described here may facilitate development of agents disrupting this interaction, which may have potential as antithrombotic drugs.

Characterization of HLA-DR- and TCR-binding residues of an immunodominant and genetically permissive peptide of the 16-kDa protein of Mycobacterium tuberculosis.

The 16-kDa protein of Mycobacterium tuberculosis represents an important antigenic target during bacillary latency and, consequently, should be considered as candidate subunit vaccine component. In this study, we have used CD4 T cell clones that recognize the peptide p91-110, an immunodominant and genetically permissive epitope, in the context of five different HLA-DR molecules and truncated and substituted variants of this peptide, to identify the minimal binding sequence (HLA-DR-binding core) and the minimal stimulatory sequence (TCR-binding core), as well as the residues that contact HLA-DR molecules and the TCR. We have found a common 9-mer sequence, spanning amino acids 93-101, as the binding core for HLA-DR1, -DR11, -DR13 and -DR7, but a longer (13-mer) sequence spanning amino acids 92-104 was required for binding to the HLA-DR15 molecules. F(93) was required for binding to all the tested HLA-DR molecules, hence allowing us to identify it as the N-terminal primary anchor residue (P1). Additionally, the binding requirements for other residues varied considerably between the tested alleles: A(94) for HLA-DR15, V(99) for HLA-DR1, -DR15, -DR11 and -DR7, R(100) for HLA-DR11 and -DR13, and L(104) for HLA-DR15. Concerning the residues of p91-110 peptide required for binding to the TCR, the pepscan analysis results would support the contention that P(-1) E(92), P6 F(98) would be important TCR contact sites because their substitutions led to full loss of T cell activation. Moreover, P8 R(100) is found to be critical residue in binding to HLA-DR11- and -DR13-restricted T cell clones, without influencing binding to the relevant HLA-DR molecule. Our results could be useful to design peptides with altered HLA anchor residues or TCR interaction sites to achieve remarkable increase in activity and to study their vaccine potential.

The monoclonal antibody mAB 1A binds to the excitation–contraction coupling domain in the II–III loop of the skeletal muscle calcium channel α 1S subunit

Interactions of the II–III loop of the voltage-gated Ca 2+ channel α 1S subunit with the Ca 2+ release channel (RyR1) are essential for skeletal-type excitation-contraction (EC) coupling. Here, we characterized the binding site of the monoclonal α 1S antibody mAB 1A and used it to probe the structure of the II–III loop in chimeras with different EC coupling properties. Phage-display epitope mapping of mAB 1A revealed a minimal consensus binding sequence X–P–X–X–D–X–P. Immunofluorescence labeling of α 1S, α 1C, α 1D, and of II–III loop chimeras expressed in dysgenic myotubes established that mAB 1A reacted specifically with amino acids 737–744 in the II–III loop of α 1S, which is within the domain (D734–L764) critical for bidirectional coupling with RyR1. Comparing mAB 1A immunoreactivity with known structural and functional properties of II–III loop chimeras in which the non-conserved skeletal residues were systematically mutated to their cardiac counterparts indicated a correlation of mAB 1A immunoreactivity and skeletal-type EC coupling.

Design, synthesis, and evaluation of peptides with estrogen-like activity.

Currently used antiestrogenic drugs against hormone-dependent breast cancer, and estrogenic drugs used in treatment of osteoporosis, are associated with risk factors. Therefore, there is a strong need to develop selective estrogen receptor modulators with better tissue selectivity. In a recent study (Peptides, 2002, Vol. 3, 573-580), we used a monoclonal antibody to estradiol (mAb-E2) to screen a phage-display peptide library. We identified a 15-mer peptide (peptide H5) that recognizes mAb-E2 (IC(50) 1 microM) and estrogen receptor (ER)alpha (IC(50) 500 microM) but not ERbeta, and displays estrogen-like activity in vitro and in vivo. In this study, we designed and prepared peptides based on peptide H5, which possess improved estrogenic activity, by evaluating their binding to mAb-E2 and to ERs. Initially, we determined the minimal binding sequence of peptide H5 capable of binding mAb-E2 and ER. Subsequently, systematic single-residue replacements of the minimal sequence, followed by multiple-residue replacements, yielded hexa- and heptapeptides with increased affinities to mAb-E2 and to ER. The most promising peptides, VSWFFE (EMP-1) and VSWFFED (EMP-2) (EMP: estrogen-mimetic peptide), bind mAb-E2 with high affinity (IC(50) of 6 and 30 nM, respectively), recognize ERs with increased affinity (IC(50) of 100 microM for ERalpha, and 100-250 microM for ERbeta), and possess estrogenic activity in vivo. The short peptides described in this study may be used as potential lead compounds for developing new ER ligands.

The Parathyroid Hormone mRNA 3′-Untranslated Region AU-rich Element Is an Unstructured Functional Element

Parathyroid hormone (PTH) gene expression is regulated post-transcriptionally by hypocalcemia and hypophosphatemia. This regulation is dependent upon binding of protective trans-acting factors to a specific element in the PTH mRNA 3'-untranslated region (UTR). We have previously demonstrated that a 63-nucleotide (nt) AU-rich PTH mRNA element is sufficient to confer regulation of RNA stability by calcium and phosphate in an in vitro degradation assay (IVDA). The 63-nt element consists of a core 26-nt minimal binding sequence and flanking regions. We have now studied the functionality of this element in HEK293 cells using reporter genes and showed that it destabilizes mRNAs for green fluorescent protein (GFP) and growth hormone, similar to its effect in the IVDA. To understand how the cis-element functions as an instability element, we have analyzed its structure by RNase H, primer extension, and computer modeling. The results indicate that the PTH mRNA 3'-UTR and in particular the region of the cis-element are dominated by significant open regions with little folded base pairing. Mutation analysis of the 26-nt core element demonstrated the importance of defined nucleotides for protein-RNA binding. In the GFP reporter system, the same mutations that prevented binding were also ineffective in destabilizing GFP mRNA in HEK293 cells. This is the first study of an AU-rich element that relates function to structure. The PTH mRNA 3'-UTR cis-acting element is an open region that utilizes the distinct sequence pattern to determine mRNA stability by its interaction with trans-acting factors.

Rational Design and Molecular Diversity for the Construction of Anti-α-Bungarotoxin Antidotes with High Affinity and In Vivo Efficiency

The structure of peptide p6.7, a mimotope of the nicotinic receptor ligand site that binds α-bungarotoxin and neutralizes its toxicity, was compared to that of the acetylcholine binding protein. The central loop of p6.7, when complexed with α-bungarotoxin, fits the structure of the acetylcholine binding protein (AChBP) ligand site, whereas peptide terminal residues seem to be less involved in toxin binding. The minimal binding sequence of p6.7 was confirmed experimentally by synthesis of progressively deleted peptides. Affinity maturation was then achieved by random addition of residues flanking the minimal binding sequence and by selection of new α-bungarotoxin binding peptides on the basis of their dissociation kinetic rate. The tetra-branched forms of the resulting high-affinity peptides were effective as antidotes in vivo at a significantly lower dose than the tetra-branched lead peptide.

Designing isoform-specific peptide disruptors of protein kinase A localization.

A kinase-anchoring proteins (AKAPs) coordinate cAMP-mediated signaling by binding and localizing cAMP-dependent protein kinase (PKA), using an amphipathic helical docking motif. Peptide disruptors of PKA localization that mimic this helix have been used successfully to assess the involvement of PKA in specific signaling pathways. However, these peptides were developed as disruptors for the type II regulatory subunit (RII) even though both RI and RII isoforms can bind to AKAPs and have discrete functions. To evaluate the effects of each localized isoform, we designed peptides that specifically bind to either RI or RII. Using a peptide array, we have defined the minimal binding sequence of dual specific-AKAP 2 (d-AKAP2), which binds tightly to both RI and RII. Side-chain requirements for affinity and isoform specificity were evaluated by using a peptide substitution array where each position along the A kinase binding domain of d-AKAP2 was substituted by the other 19 l-amino acids. This array comprises 513 single-site substitution analogs of the d-AKAP2 sequence. Peptides containing single and multiple mutations were evaluated in a quantitative fluorescence binding assay and a cell-based colocalization assay. This strategy has allowed us to design peptides with high affinity (K(D) = 1-2 nM) and high specificity for RIalpha versus RIIalpha. These isoform-specific peptides will be invaluable tools to evaluate functional differences between localized RI and RII PKA and are RIalpha-specific disruptors. This array-based analysis also provides a foundation for biophysical analysis of this docking motif.