Mapping of a putative surface-binding site of human coagulation factor XII

We have localized the binding epitope(s) of two murine monoclonal antibodies (B7C9 and P5-2-1) that were shown previously to inhibit the activation of human coagulation factor XII by negatively charged surfaces. A factor XII cDNA expression library in lambda gt11 was screened with antibody B7C9, and 16 immunoreactive bacteriophage were isolated. Fusion proteins from each of the recombinant phage were reactive with both monoclonal antibodies. Two of the phage cDNA inserts were found to code for amino acid residues -6-+31 and +1-+47 of factor XII, respectively, thereby defining the limits of the antigenic peptide to amino acids +1-+31. Each of the remaining 14 recombinant phage contained longer factor XII cDNA inserts that included sequences coding for the amino-terminal 31 amino acid residues. These results were confirmed by direct binding of antibody B7C9 to synthetic peptides containing amino acids 1-14 and 1-28 of factor XII. Further experiments with a set of nested peptides also indicated that amino acid residues 1-4 were essential but not sufficient for binding of B7C9 to the peptides. Hydrophobicity analysis of the amino-terminal region of plasma factor XII revealed a highly hydrophilic region between amino acid residues 5 and 15 that contained positively charged lysine residues at positions 8, 11, and 13. We conclude that a major epitope(s) recognized by monoclonal antibodies B7C9 and P5-2-1 is present in the amino-terminal 28 amino acids of factor XII. It is proposed that binding of these antibodies to factor XII blocks interaction of the positively charged region between residues 5 and 15 with negatively charged surfaces, thereby inhibiting activation.     

Novel cyclization chemistry especially suited for biologically derived, unprotected peptides.

A novel method is described for the cyclization of peptides--or segments of polypeptides--which requires a free N-terminal alpha-amino group and a distal amino acid residue containing a nucleophilic side chain. The reaction is conducted in two steps, both in the aqueous phase. The first step involves acylation of the N-terminal alpha-amino group with iodoacetic anhydride at pH 6. This acylation reaction has greater than 90% specificity for peptide alpha-amino groups and gives no alkylation of Arg, His, Lys or Met by the iodoacetate side product (R. Wetzel et al., Bioconjugate Chem., 1, 114-122, 1990). In the second step, the acylation reaction mixture or the isolated iodoacetyl-peptide is incubated at room temperature to give the cyclic peptide formed by reaction of the nucleophilic side chain with the iodoacetyl moiety. The pH dependence of the cyclization reaction by Met, Lys, Arg or His is consistent with the pKa of the nucleophilic side chain. Thus, peptides containing Met plus other nucleophilic amino acids should preferentially cyclize via Met at low pH. In this paper, preparation of cyclic peptides containing 3-6 amino acids is described; the full range of ring sizes and sequences which can undergo this cyclization has not been further explored. Preliminary results suggest that this method is also fairly general with respect to the amino acid sequence being cyclized. The reaction appears to be particularly suited for cyclization via Lys and Met side chains. All of the cyclized products are sufficiently stable for many biological applications.(ABSTRACT TRUNCATED AT 250 WORDS)     

The tryptic peptides of rabbit muscle triose phosphate isomerase

1. The peptides obtained by tryptic digestion of S-[(14)C]carboxymethylated rabbit muscle triose phosphate isomerase have been studied. 2. The first step in the fractionation of the tryptic digest was gel filtration on coupled columns of Sephadex G-25 and G-50. Further fractionation was carried out by paper electrophoresis and paper chromatography. 3. The digest contained 26 peptides and three free amino acids. The sizes of the peptides ranged from two to 29 residues. 4. The sequences of the peptides have been determined. 5. The length of the polypeptide chains is about 250 amino acid residues. 6. The variant sequences encountered were due to partial deamidation; this may be one of the reasons for multiple forms of the enzyme. 7. The chicken and rabbit enzymes are compared. 8. Detailed evidence for the sequences of the tryptic peptides has been deposited as Supplementary Publication SUP 50024 at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1973) 131, 5.     

Synthesis and DNA nicking studies of a novel cyclic peptide: cyclo[Lys-Trp-Lys-Ahx-].

Two novel cyclic tetrapeptides: cyclo[Lys-Tyr-Lys-Ahx-] 7a and cyclo[Lys-Trp-Lys-Ahx-] 7b were synthesized by coupling protected amino acid in solution and the subsequent cyclization effected by the pentafluorophenyl ester method as described in previous papers. These cyclic peptides were designed and synthesized to study their interaction with DNA, based on previous reports that linear peptides Lys-Tyr-Lys and Lys-Trp-Lys could bind to various forms of DNA and cleaved supercoiled DNA at apurinic sites. Ethidium bromide displacement assay showed that the apparent DNA binding constant of linear Lys-Tyr-Lys and cyclic peptide 7a are far below 1 x 10(3) M(-1), whereas those of cyclic peptide 7b and linear Lys-Trp-Lys are 1.9 x 10(4) M(-1) and 9.5 x 10(3) M(-1), respectively. Kinetic studies using agarose gel electrophoresis showed that cyclic peptide 7b and Lys-Trp-Lys possessed DNA nicking activity on natural supercoiled phi X174 DNA with nicking rate of 50.7 and 75.6 pM min(-1) at 65 degrees C, respectively, whereas cyclic peptide 7a and linear Lys-Tyr-Lys were devoid of the corresponding activity. The DNA nicking rate increased significantly with increase in reaction temperature. At reaction temperatures lower than 65 degrees C, the DNA nicking rate of cyclic peptide 7b exceeded that of linear Lys-Trp-Lys. The addition of 1 microM ferrous ion did not give significant enhancement effect on the DNA nicking rate by the peptides. UV irradiation gave a marked rate enhancement on the DNA nicking rate of linear Lys-Trp-Lys and a moderate enhancement on the DNA nicking rate of cyclic peptide 7b.     

Identification of an ADAMTS-4 cleavage motif using phage display leads to the development of fluorogenic peptide substrates and reveals matrilin-3 as a novel substrate

ADAMTS-4 and ADAMTS-5 are aggrecanases responsible for the breakdown of cartilage aggrecan in osteoarthritis. Multiple ADAMTS-4 cleavage sites have been described in several matrix proteins including aggrecan, versican, and brevican, but no concise predictive cleavage motif has been identified for this protease. By screening a 13-mer peptide library with a diversity of 10(8), we have identified the ADAMTS-4 cleavage motif E-(AFVLMY)-X(0,1)-(RK)-X(2,3)-(ST)-(VYIFWMLA), with Glu representing P1. Several 13-mer peptides containing this motif, including DVQEFRGVTAVIR and HNEFRQRETYMVF, were shown to be substrates for ADAMTS-4. These peptides were found to be specific substrates for ADAMTS-4 as they were not cleaved by ADAMTS-5. Modification of these peptides with donor (6-FAM) and acceptor (QSY-9) molecules resulted in the development of fluorescence-based substrates with a Km of approximately 35 microM. Furthermore, the role of Glu at P1 and Phe at P1' in binding and catalysis was studied by exploring substitution of these amino acids with the D-isomeric forms. Substitution of P1 with dGlu was tolerable for binding, but not catalysis, whereas substitution of P1' with dPhe precluded both binding and catalysis. Similarly, replacement of Glu with Asp at P1 abolished recognition and cleavage of the peptide. Finally, BLAST results of the ADAMTS-4 cleavage motif identified matrilin-3 as a new substrate for ADAMTS-4. When tested, recombinant ADAMTS-4 effectively cleaved intact matrilin-3 at the predicted motif at Glu435/Ala436 generating two species of 45 and 5 kDa.     

Synthesis and biological evaluation of cyclic and branched peptide analogues as ligands for cholecystokinin type 1 receptor

A library of cyclic CCK8 analogues, containing unnatural amino acids in the peptide sequence, is prepared using solid-phase synthesis. The structure of these cyclic peptides is based on a previously synthesised compound, cyclo-CCK8, selective for CCK(1) receptor. Structure-activity investigations are performed by evaluating the binding properties of the new analogues. In particular, the binding ability of the cyclic CCK8 analogues is tested by nuclear medicine studies on cell line transfected with CCK(1) receptor. Compounds named cyclo-A4-cyclo-A7 show binding constant in the range 6.0-8.0 microM, with an improved affinity over the previous described cyclo-CCK8, but almost comparable IC(50) values among new analogues towards CCK(1) were obtained.     

Determination of the binding specificity of the SH2 domains of protein tyrosine phosphatase SHP-1 through the screening of a combinatorial phosphotyrosyl peptide library

A method for the rapid identification of high-affinity ligands to Src homology-2 (SH2) domains is reported. A phosphotyrosyl (pY) peptide library containing completely randomized residues at positions -2 to +3 relative to the pY was synthesized on TentaGel resin, with a unique peptide sequence on each resin bead (total 2.5 x 10(6) different sequences). The library was screened against the biotinylated N- and C-terminal SH2 domains of protein tyrosine phosphatase SHP-1, and the beads that carry high-affinity ligands of the SH2 domains were identified using an enzyme-linked assay involving a streptavidin-alkaline phosphatase conjugate. Peptide ladder sequencing of the selected beads using matrix-assisted laser desorption ionization mass spectrometry revealed consensus sequences for both SH2 domains. The N-terminal SH2 domain strongly selects for peptides with a leucine at the -2 position; at the C-terminal side of the pY residue, it can recognize two distinct classes of peptides with consensus sequences of LXpY(M/F)X(F/M) and LXpYAXL (X = any amino acid), respectively. The C-terminal SH2 domain exhibits almost exclusive selectivity for peptides of the consensus sequence, (V/I/L)XpYAX(L/V). Several representative sequences selected from the library were individually synthesized and tested for binding to the SH2 domains by surface plasmon resonance and for their ability to stimulate the catalytic activity of SHP-1. Both experiments have demonstrated that the selected peptides are capable of binding to the SH2 domains with dissociation constants (K(D)) in the low micromolar range.     

传染性法氏囊病病毒五个抗原表位短肽的鉴定与序列分析

以5株传染性法氏囊病病毒(Infectious bursal disease virus,IBDV)单克隆抗体HNF1、HNF7、B34、2B1和2G8作为筛选分子,对噬菌体展示12肽库进行3轮"吸附-洗脱-扩增"淘洗,从每株单克隆抗体筛选到的噬菌斑中随机挑取12个单克隆蓝色噬菌斑,合计60个,用间接ELISA检测,A值大于1.00;用竞争抑制ELISA分析,单克隆抗体和IBDV抗原均能竞争抑制筛选12肽与固相包被单克隆抗体的反应,抑制率大于40%,表明在该12肽内含有IBDV抗原表位。选取35个单克隆噬菌斑,测定噬菌体gIII部分基因的核苷酸序列,确定了这5个含有不同IBDV抗原表位12肽的核苷酸和氨基酸序列。进一步将其与GenBank中IBDV基因组编码蛋白的氨基酸序列进行比较,发现2B1筛选肽有4个连续氨基酸残基Leu-Ala-Ser-Pro与IBDV基因组A片段编码多聚蛋白的第536-599氨基酸残基一致,推测2B1为线性表位;而HNF1、HNF7、B34和2G8筛选肽均没找到有3个以上连续氨基酸残基与IBDV蛋白序列相同之处,推测可能是构象依赖性表位。     

Bradykinins and their precursor cDNAs from the skin of the fire-bellied toad (Bombina orientalis)

Bradykinin and (Thr(6))-bradykinin have been identified in the defensive skin secretion of the fire-bellied toad, Bombina orientalis. The homologous cDNAs for both peptides were cloned from a skin library using a 3'- and 5'-RACE strategy. Kininogen-1 (BOK-1) contained an open-reading frame of 167 amino acid residues encoding four repeats of bradykinin, and kininogen-2 (BOK-2) contained an open-reading frame of 161 amino acid residues encoding two repeats of (Thr(6))-bradykinin. Alignment of both precursor nucleotide and amino acid sequences revealed a high degree of structural similarity. These amphibian skin kininogens/preprobradykinins are not biologically analogous to mammalian kininogens.     

Structure-activity relationship and metabolic stability studies of backbone cyclization and N-methylation of melanocortin peptides

Backbone cyclization (BC) and N-methylation have been shown to enhance the activity and/or selectivity of biologically active peptides and improve metabolic stability and intestinal permeability. In this study, we describe the synthesis, structure-activity relationship (SAR) and intestinal metabolic stability of a backbone cyclic peptide library, BL3020, based on the linear alpha-Melanocyte stimulating hormone analog Phe-D-Phe-Arg-Trp-Gly. The drug lead, BL3020-1, selected from the BL3020 library (compound 1) has been shown to inhibit weight gain in mice following oral administration. Another member of the BL3020 library, BL3020-17, showed improved biological activity towards the mMC4R, in comparison to BL3020-1, although neither were selective for MC4R or MC5R. N-methylation, which restrains conformational freedom while increasing metabolic stability beyond that which is imparted by BC, was used to find analogs with increased selectivity. N-methylated backbone cyclic libraries were synthesized based on the BL3020 library. SAR studies showed that all the N-methylated backbone cyclic peptides demonstrated reduced biological activity and selectivity for all the analyzed receptors. N-methylation of active backbone cyclic peptides destabilized the active conformation or stabilized an inactive conformation, rendering the peptides biologically inactive. N-methylation of backbone cyclic peptides maintained stability to degradation by intestinal enzymes.     

Automatic Edman microsequencing of peptides containing multiple unnatural amino acids

It is now routine using automatic Edman microsequencing to determine the primary structure of peptides or proteins containing natural amino acids; however, a deficiency in the ability to readily sequence peptides containing unnatural amino acids remains. With the advent of synthetic peptide chemistry, combinatorial chemistry, and the large number of commercially available unnatural amino acids, there is a need for efficient and accurate structure determination of short peptides containing many unnatural amino acids. In this study, 35 commercially available alpha-unnatural amino acids were selected to determine their elution profile on an ABI protein sequencer. Using a slightly modified gradient program, 19 of these 35 PTH amino acids can be readily resolved and distinguished from common PTH amino acids at low picomole levels. These unnatural amino acids in conjunction with the 20 natural amino acids can be used as building blocks to construct peptide libraries, and peptide beads isolated from these libraries can be readily microsequenced. To demonstrate this, we synthesized a simple tripeptide "one-bead one-compound" combinatorial library containing 14 unnatural and 19 natural amino acids and screened this library for streptavidin-binding ligands. Microsequencing of the isolated peptide-beads revealed the novel motif Bpa-Phe(4-X)-Aib, wherein X = H, OH, and CH3.     

Recognition of a flipped base in a hairpinloop DNA by a small peptide

Two tiny hairpin DNAs, CORE (dAGGCTTCGGCCT) and AP2 (dAGGCTXCGGCCT; X: abasic nucleotide), fold into almost the same tetraloop hairpin structure with one exception, that is, the sixth thymine (T6) of CORE is exposed to the solvent water (Kawakami, J. et al., Chem. Lett. 2001, 258-259). In the present study, we selected small peptides that bind to CORE or AP2 from a combinatorial pentapeptide library with 2.5 x 10(6) variants. On the basis of the structural information, the selected peptide sequences should indicate the essential qualifications for recognition of the hairpin loop DNA with and without a flipped base. In the selected DNA binding peptides, aromatic amino acids such as histidine for CORE and glutamine/aspartic acid for AP2 were found to be abundant amino acids. This amino acid preference suggests that CORE-binding peptides use pi-pi stacking to recognize the target while hydrogen bonding is dominant for AP2-binding peptides. To investigate the binding properties of the selected peptide to the target, surface plasmon resonance was used. The binding constant of the interaction between CORE and a CORE-binding peptide (HWHHE) was about 1.1 x 10(6) M(-1) at 25 degrees C and the resulting binding free energy change at 25 degrees C (DeltaG degrees (25)) was -8.2 kcal mol(-1). The binding of the peptide to AP2 was also analyzed and the resulting binding constant and DeltaG degrees (25) were about 4.2 x 10(4) M(-1) and -6.3 kcal mol(-1), respectively. The difference in the binding free energy changes (DeltaDeltaG degrees (25)) of 1.9 kcal mol(-1) was comparable to the values reported in other systems and was considered a consequence of the loss of pi-pi stacking. Moreover, the stabilization effect by stacking affected the dissociation step as well as the association step. Our results suggest that the existence of an aromatic ring (T6 base) produces new dominant interactions between peptides and nucleic acids, although hydrogen bonding is the preferable mode of interaction in the absence of the flipping base. These findings regarding CORE and AP2 recognition are expected to give useful information in the design of novel artificial DNA binding peptides.     

Identification of calmodulin isoform-specific binding peptides from a phage-displayed random 22-mer peptide library

Plants express numerous calmodulin (CaM) isoforms that exhibit differential activation or inhibition of CaM-dependent enzymes in vitro; however, their specificities toward target enzyme/protein binding are uncertain. A random peptide library displaying a 22-mer peptide on a bacteriophage surface was constructed to screen peptides that specifically bind to plant CaM isoforms (soybean calmodulin (ScaM)-1 and SCaM-4 were used in this study) in a Ca2+-dependent manner. The deduced amino acid sequence analyses of the respective 80 phage clones that were independently isolated via affinity panning revealed that SCaM isoforms require distinct amino acid sequences for optimal binding. SCaM-1-binding peptides conform to a 1-5-10 ((FILVW)XXX(FILV) XXXX(FILVW)) motif (where X denotes any amino acid), whereas SCaM-4-binding peptide sequences conform to a 1-8-14 ((FILVW)XXXXXX(FAILVW)XXXXX(FILVW)) motif. These motifs are classified based on the positions of conserved hydrophobic residues. To examine their binding properties further, two representative peptides from each of the SCaM isoform-binding sequences were synthesized and analyzed via gel mobility shift assays, Trp fluorescent spectra analyses, and phosphodiesterase competitive inhibition experiments. The results of these studies suggest that SCaM isoforms possess different binding sequences for optimal target interaction, which therefore may provide a molecular basis for CaM isoform-specific function in plants. Furthermore, the isolated peptide sequences may serve not only as useful CaM-binding sequence references but also as potential reagents for studying CaM isoform-specific function in vivo.     

Further biochemical and physicochemical characterization of minor disulfide-bonded (type IX) collagen, extracted from foetal calf cartilage

Minor disulfide-bonded collagen (previously termed X1-X7 and now called type IX collagen) was isolated from foetal calf cartilage after pepsin treatment. At least three native fractions, containing, respectively, the X1X2X3, X4, and X5X6X7 chains, were separated; and from further biochemical and physicochemical experiments (differential scanning calorimetry, electrical birefringence, rotary shadowing), we propose a tentative model for their organization within a parent molecule. X1 and X2 are molecules composed of three chains of apparent Mr 62,000 and 50,000 linked by interchain disulfide bonds and containing pepsin-sensitive regions. The cleavage of at least three of these sites, present within X2, gives rise to the X3 and X5X6X7 fractions composed of molecules 80-100 nm and 40-55 nm in length, respectively. The X5X6X7 fraction is not digested by pepsin at 30 degrees C owing to its high thermal stability (certainly explained by its high hydroxyproline + proline content). This organization is in good accordance with that proposed for chicken cartilage type IX collagen; differences could only exist in the number and (or) the location of the pepsin-sensitive sites.     

Structures of the contryphan family of cyclic peptides. Role of electrostatic interactions in cis-trans isomerism

The contryphan family of cyclic peptides, isolated recently from various species of cone shell, has the conserved sequence motif NH(3)(+)-X(1)COD-WX(5)PWC-NH(2), where X(1) is either Gly or absent, O is 4-trans-hydroxyproline, and X(5) is Glu, Asp, or Gln. The solution structures described herein of two new naturally occurring contryphan sequences, contryphan-Sm and des[Gly1]-contryphan-R, are similar to those of contryphan-R, the structure of which has been determined recently [Pallaghy et al. (1999) Biochemistry 38, 11553-11559]. The (1)H NMR chemical shifts of another naturally occurring peptide, contryphan-P, indicate that it also adopts a similar structure. All of these contryphans exist in solution as a mixture of two conformers due to cis-trans isomerization about the Cys2-Hyp3 peptide bond. The lower cis-trans ratio for contryphan-Sm enabled elucidation of the 3D structure of both its major and its minor forms, for which the patterns of (3)J(H)(alpha)(HN) coupling constants are very different. As with contryphan-R, the structure of the major form of contryphan-Sm (cis Cys2-Hyp3 peptide bond) contains an N-terminal chain reversal and a C-terminal type I beta-turn. The minor conformer (trans peptide bond) forms a hairpin structure with sheetlike hydrogen bonds and a type II beta-turn, with the D-Trp4 at the 'Gly position' of the turn. The ratio of conformers arising from cis-trans isomerism around the peptide bond preceding Hyp3 is sensitive to both the amino acid sequence and the solution conditions, varying from 2.7:1 to 17:1 across the five sequences. The sequence and structural determinants of the cis-trans isomerism have been elucidated by comparison of the cis-trans ratios for these peptides with those for contryphan-R and an N-acetylated derivative thereof. The cis-trans ratio is reduced for peptides in which either the charged N-terminal ammonium or the X(5) side-chain carboxylate is neutralized, implying that an electrostatic interaction between these groups stabilizes the cis conformer relative to the trans. These results on the structures and cis-trans equilibrium of different conformers suggest a paradigm of 'locally determined but globally selected' folding for cyclic peptides and constrained protein loops, where the series of stereochemical centers in the loop dictates the favorable conformations and the equilibrium is determined by a small number of side-chain interactions.     

Two novel Bv8-like peptides from skin secretions of the toad Bombina maxima

Two novel bioactive peptides were purified from skin secretions of the toad Bombina maxima. The partial N-terminal sequences of these two peptides were determined by automated Edman degradation. This allowed the cloning of full-length cDNAs encoding these two peptides from a cDNA library prepared from the toad skin. The deduced complete amino acid sequences indicate that both peptides are composed of 77 amino acids. A FASTA search in the databanks revealed that they exhibit 86-91% sequence identity with Bv8, a peptide originally isolated from skin secretions of Bombina variegata. They were thus named as Bv8-like peptide 1 (Bv8-LP1) and Bv8-like peptide 2 (Bv8-LP2), respectively. Sequence differences between Bv8-LP1 and 2 were due to six amino acid substitutions at positions 6, 11, 23, 24, 62 and 63. Bv8-LP1 and 2 differed from Bv8 with eleven and seven amino acid substitutions, respectively. Like Bv8, Bv8-LP1 and 2 possessed contractile activity on isolated guinea pig ileum. Additionally, they stimulated contraction of rabbit aortic rings in a dose-dependent manner at nanomolar concentrations.     

Improvement of cyclic decapeptides against plant pathogenic bacteria using a combinatorial chemistry approach

Cyclic decapeptides were developed based on the previously reported peptide c(LysLeuLysLeuLysPheLysLeuLysGln). These compounds were active against the economically important plant pathogenic bacteria Erwinia amylovora, Pseudomonas syringae and Xanthomonas vesicatoria. A library of 56 cyclic decapeptides was prepared and screened for antibacterial activity and eukaryotic cytotoxicity, and led to the identification of peptides with improved minimum inhibitory concentration (MIC) against P. syringae (3.1-6.2 microM) and X. vesicatoria (1.6-3.1 microM). Notably, peptides active against E. amylovora (MIC of 12.5-25 microM) were found, constituting the first report of cyclic peptides with activity towards this bacteria. A second library based on the structure c(X(1)X(2)X(3)X(4)LysPheLysLysLeuGln) with X being Lys or Leu yielded peptides with optimized activity profiles. The activity against E. amylovora was further improved (MIC of 6.2-12.5 microM) and the best peptides displayed a low eukaryotic cytotoxicity at concentrations 30-120 times higher than the MIC values. A design of experiments permitted to define rules for high antibacterial activity and low cytotoxicity, being the main rule X(2) not equal X(3), and the secondary rule X(4)=Lys. The best analogs can be considered as good candidates for the development of effective antibacterial agents for use in plant protection.     

Isolation of a highly specific ligand for the alpha 5 beta 1 integrin from a phage display library

Our previous studies showed that the alpha 5 beta 1 integrin selects cysteine pair-containing RGD peptides from a phage display library based on a random hexapeptide. We have therefore searched for more selective peptides for this integrin using a larger phage display library, where heptapeptides are flanked by cysteine residues, thus making the inserts potentially cyclic. Most of the phage sequences that bound to alpha 5 beta 1 (69 of 125) contained the RGD motif. Some of the heptapeptides contained an NGR motif. As the NGR sequence occurs in the cell-binding region of the fibronectin molecule, this sequence could contribute to the specific recognition of fibronectin by alpha 5 beta 1. Selection for high affinity peptides for alpha 5 beta 1 surprisingly yielded a sequence RRETAWA that does not bear obvious resemblance to known integrin ligand sequences. The synthetic cyclic peptide GACRRETAWACGA (*CRRETAWAC*) was a potent inhibitor of alpha 5 beta 1-mediated cell attachment to fibronectin. This peptide is nearly specific for the alpha 5 beta 1 integrin, because much higher concentrations were needed to inhibit the alpha v beta 1 integrin, and there was no effect on alpha v beta 3- and alpha v beta 5-mediated cell attachment to vitronectin. The peptide also did not bind to the alpha IIb beta 3 integrin. *CRRETAWAC* appears to interact with the same or an overlapping binding site in alpha 5 beta 1 as RGD, because cell attachment to *CRRETAWAC* coated on plastic was divalent cation dependent and could be blocked by an RGD-containing peptide. These results reveal a novel binding specificity in the alpha 5 beta 1 integrin.     

Identification of calmodulin-binding peptide consensus sequences from a phage-displayed random peptide library

The calcium-binding protein, calmodulin (CaM), was used to screen a phage library displaying random peptides 26 amino acids (aa) in length. Twenty CaM-binding peptides were identified, 17 of which contained one of three consensus sequence motifs: + W-OλR, WRAAV or WRXXAAAL, where +, -, O,λ and X are positively charged, negatively charged, hydrophobic, leucine or valine, and any residue, respectively. The Trp residue in these motifs is located within 14 aa of the N-terminus of the displayed peptide. Previous studies [Dedman et al., J. Biol. Chem. 268 (1993) 23025–23030] using a library displaying random peptides 15 aa in length identified CaM-binding peptides which contained a Trp-Pro dipeptide motif. These results suggest that the type of CaM-binding motif identified can vary between different types of combinatorial peptides