Alignment Of Peptides Research Articles

Protein-like molecular architecture: biomaterial applications for inducing cellular receptor binding and signal transduction.

The development of biomaterials with desirable biocompatibility has presented a difficult challenge for tissue engineering researchers. First and foremost, materials themselves tend to be hydrophobic and/or thrombogenic in nature, and face compatibility problems upon implantation. To mediate this problem, researchers have attempted to graft protein fragments onto biomaterial surfaces to promote endothelial cell attachment and minimize thrombosis. We envisioned a novel approach, based on the capability of biomolecules to self-assemble into well-defined and intricate structures, for creating biomimetic biomaterials that promote cell adhesion and proliferation. One of the most intriguing self-assembly processes is the folding of peptide chains into native protein structures. We have developed a method for building protein-like structural motifs that incorporate sequences of biological interest. A lipophilic moiety is attached onto a N alpha-amino group of peptide chain, resulting in a "peptide-amphiphile." The alignment of amphiphilic compounds at the lipid-solvent interface is used to facilitate peptide alignment and structure initiation and propagation, while the lipophilic region absorbs to hydrophobic surfaces. Peptide-amphiphiles containing potentially triple-helical or alpha-helical structural motifs have been synthesized. The resultant head group structures have been characterized by CD spectroscopy and found to be thermally stable over physiological temperature ranges. Triple-helical peptide-amphiphiles have been applied to studies of surface modification and cell receptor binding. Cell adhesion and spreading was promoted by triple-helical peptide-amphiphiles. Cellular interaction with the type IV collagen sequence alpha 1(IV) 1263-1277 increased signal transduction, with both the time and level of induction dependent upon triple-helical conformation. Collectively, these results suggest that peptide-amphiphiles may be used to form stable molecular structure on biomaterial surfaces that promote cellular activities and improve biocompatibility.

Proteinlike molecular architecture: Biomaterial applications for inducing cellular receptor binding and signal transduction

The development of biomaterials with desirable biocompatibility has presented a difficult challenge for tissue engineering researchers. First and foremost, materials themselves tend to be hydrophobic and/or thrombogenic in nature, and face compatibility problems upon implantation. To mediate this problem, researchers have attempted to graft proteins or protein fragments onto biomaterial surfaces to promote endothelial cell attachment and minimize thrombosis. We envisioned a novel approach, based on the capability of biomolecules to self-assemble into well-defined and intricate structures, for creating biomimetic biomaterials that promote cell adhesion and proliferation. One of the most intriguing self-assembly processes is the folding of peptide chains into native protein structures. We have developed a method for building proteinlike structural motifs that incorporate sequences of biological interest. A lipophilic moiety is attached onto an Nα-amino group of a peptide chain, resulting in a “peptide-amphiphile.” The alignment of amphiphilic compounds at the lipid-solvent interface is used to facilitate peptide alignment and structure initiation and propagation, while the lipophilic region adsorbs to hydrophobic surfaces. Peptide-amphiphiles containing potentially triple-helical or α-helical structural motifs have been synthesized. The resultant head group structures have been characterized by CD spectroscopy and found to be thermally stable over physiological temperature ranges. Triple-helical peptide-amphiphiles have been applied to studies of surface modification and cell receptor binding. Cell adhesion and spreading was promoted by triple-helical peptide-amphiphiles. Cellular interaction with the type IV collagen sequence α1(IV) 1263–1277 increased signal transduction, with both the time and level of induction dependent upon triple-helical conformation. Collectively, these results suggest that peptide-amphiphiles may be used to form stable molecular structures on biomaterial surfaces that promote cellular activities and improve biocompatibility. © 1998 John Wiley & Sons, Inc. Biopoly 47: 143–151, 1998

The Two Subunits of a Phospholipase A2 Inhibitor from the Plasma of Thailand Cobra Having Structural Similarity to Urokinase-Type Plasminogen Activator Receptor and Ly-6 Related Proteins

Amino acid sequences of the two subunits (31-kDa and 25-kDa subunits) of a phospholipase A2 (PLA2) inhibitory protein, purified from the blood plasma of Thailand cobra Naja naja kaouthia, were determined by alignment of peptides obtained by lysyl endopeptidase, staphylococcal V8 protease and endoproteinase Asp-N digestions. The respective subunits were composed of 188 and 185 amino acid residues, and the former contained one asparagine-linked sugar chain at the position 157. There was 29% identity between 31-kDa and 25-kDa subunits. The analysis of internal homology in each sequence of the two subunits revealed the existence of two repeats of approximately 90 amino acid residues. These sequence units were found to be significantly homologous to those of urokinase-type plasminogen activator receptor and of Ly-6 related proteins, such as Ly-6A/E, Ly-6C, ThB, and CD59 antigens.

Ligand motifs of HLA-DRB5*0101 and DRB1*1501 molecules delineated from self-peptides.

Antigenic peptides are presented to CD4+ T cells by MHC class II molecules via a highly polymorphic peptide-binding groove. The two HLA-DR alleles isotypically expressed on HLA-DR15Dw2-positive cells, DRB1*1501 (DR2b) and DRB5*0101 (DR2a) molecules, show a number of differences in polymorphic residues of the beta-chain, including the Gly-Val-dimorphism at position beta 86. Therefore, different requirements for interaction of peptides with these alleles must be expected. In this study, naturally processed self-peptides were eluted from purified HLA-DR15Dw2 molecules and related to DRB1*1501 or DRB5*0101 molecules by binding assays. An alignment of self-peptides and foreign peptides allowed the delineation of putative anchor motifs. N- and C-terminally truncated and alanine-substituted derivatives of the DR15Dw2 restricted myelin basic protein epitope MBP(85-105) confirmed their validity. Thus, DRB5*0101 requires a bulky hydrophobic residue (F or Y) at position i as a primary anchor, and Q or an aliphatic residue, such as V, I, or M, at position i + 3; positively charged residues at positions i + 7 and i + 8 are secondary anchors. For DRB1*1501, a nonaromatic, hydrophobic anchor (L, V, or I) at position i is supplemented by a bulky hydrophobic residue (F or Y) at position i + 3 as primary anchor; an additional hydrophobic side chain represented by M, I, V, or F occurs at position i + 6. Therefore, MBP(85-105) seems to contain two MHC interaction sites for DRB1*1501 and DRB5*0101, respectively, that may contribute to its immunodominance. Because HLA-DR15 Dw2 is associated with susceptibility to develop multiple sclerosis, the delineation of ligand motifs of the two DR2 alleles may help to study the interaction between potential autoantigenic peptides and these molecules in the future.

An algorithm to determine protein sequence alignment by utilizing data obtained from a peptide mixture and individual peptides.

With the aim of limiting peptide purification steps and unambiguously ascertaining protein sequences, we have designed and implemented on a personal computer an algorithm to determine sequence alignment by utilizing data obtained from automatic Edman degradation performed on a single peptide mixture and individual peptides. The protein under study is digested by two different hydrolysis methods and fragments are just isolated from one mixture and sequenced, while the second mixture is submitted unfractionated to sequence analysis. The algorithm provides for the exact alignment of the individual peptides using the mixture data for the overlapping. We report an example of application of this approach by utilizing experimental data obtained from a protein of known sequence.

A model for the calmodulin—peptide complex based on the troponin C crystal packing and its similarity to the NMR structure of the calmodulin—myosin light chain kinase peptide complex

In the crystal structure of troponin C, the holo C-domain is bound in a head-to-tail fashion to the A-helix of the apo N-domain of a symmetry-related molecule. Using this interaction, we have proposed a model for the calmodulin-peptide complex. We find that the interaction of the C-domain with the A-helix is similar to that observed in the NMR structure of the calmodulin-myosin light chain kinase (MLCK) peptide complex. This similarity in binding has enabled us to make a precise sequence alignment of the target peptides in the calmodulin-binding cleft and to rationalize the amino acid sequence-dependent binding strengths of various peptides. Our model differs from that proposed by Strynadka and James (Proteins Struct. Funct. Genet. 7, 234-248, 1990) in that the peptides are rotated by 100 degrees in the calmodulin binding cleft.

Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.

Plasma lipid transport in the preruminant calf, Bos spp: Primary structure of bovine apolipoprotein A-I

The preruminant calf (Bos spp.) is a model of considerable interest with regard to hepatic and intestinal lipoprotein metabolism (Bauchart et al., J. Lipid Res. (1989) 30, 1499–1514 and Laplaud et al., J. Lipid Res. (1990) 31, 1781–1792). As a preliminary step towards future experiments dealing with HDL metabolism in the calf, we have purified apoA-I from this animal and determined its complete amino acid sequence. Thus, approx. 10% of calf apoA-I was shown to contain a propeptide, with the sequence Arg-His-Phe-Trp-Gln-Gln. Enzymatic cleavage of apoA-I resulted in 10 proteolytic peptides. The complete apoA-I sequence was obtained after alignment of peptides on the basis of their homologies with those from rabbit apoA-I. Thus calf apoA-I consists of 241 amino acid residues, and exhibits high sequence homology with all mammalian apoA-I's studied to date. The bovine protein contained 10 hydrophobic amphipathic helical regions, occurring between residues 43–64, 65–86, 87–97, 98–119, 120–141, 142–163, 164–184, 185–206, 207–217 and 218–241. A computer-constructed phylogenetic tree showed that bovine apoA-I was more closely related to its dog counterpart, including the presence of a single methionine, than to the corresponding macaque and human proteins. Comparative predictions of the respective antigenic structures of human and bovine apoA-I's using the Hopp-Woods algorithm indicated similar positions for all 13 detectable antigenic sites, among which 7 were of identical, or closely related, amino acid composition. This finding was confirmed by demonstration of partial immunological identity between the two proteins upon immunodiffusion analysis, a result obtained using a monospecific rabbit antiserum against bovine apoA-I. Finally, comparison of sequence homology between bovine apoA-I and the lecithin : cholesterol acyl transferase (LCAT) activating region of human apoC-I suggests that several LCAT activating domains may be present in calf apoA-I.

THE PRIMARY STRUCTURE OF ARROWHEAD PROTEINASE INHIBITOR

After the reduction and carboxymethylation of disulfide bonds, arrowhead proteinase inhibitors A and B were cleaved either by proteinases or by cyanogen bromide, the fractionated and purified peptides were then subjected to sequencing by a gas phase automatic sequencer, the primary structures were completed by the alignment of the peptides sequenced with overlapping peptides. Both inhibitors A and B consist of 150 amino acid residues with three pairs of disulfide bonds, share 90% homology in structure, and are markedly different from all other Ser proteinase inhibitors so far known. Hence, the arrowhead inhibitor may belong to a new inhibitor family. Based on their structure characteristics, it was deduced that both their two reactive sites might be located in the positions of Lys-Ser (45-46) and Arg-Tyr-Lys (77-79), respectively. Among 13 mutated residues in inhibitors A and B, the substitution of residue Arg in position 87 of inhibitor B for residue Leu in A might be the main cause of leading to difference in their inhibitory activities.

A new procedure for peptide alignment in protein sequence determination using fast atom bombardment mass spectral data

A computer program allowing the correct alignment of peptides generated by a first cleaving agent during protein sequence determination studies has been developed. The program elaborates data obtained from fast atom bombardment mass spectrometric analysis of different digests of the protein. The recorded mass values are used to identify peptides in these digests that overlap peptides from the first cleavage, thus making it possible to establish unambiguously the correct order of these peptides in the protein chain. This procedure has been tested on a model protein by reconstructing the complete sequence of human beta-globin chain, determining the correct alignment of 14 tryptic peptides.

Amino acid sequences of the two subunits of a phospholipase A2 inhibitor from the blood plasma of Trimeresurus flavoviridis. Sequence homologies with pulmonary surfactant apoprotein and animal lectins.

Phospholipase A2 inhibitor (PLI), purified from the blood plasma of the Habu snake (Trimeresurus flavoviridis), was separated into two distinct subunits, PLI-A and PLI-B. These subunits were shown to be glycoproteins with molecular weights of around 21,000-22,000. When they were deglycosylated chemically with trifluoromethanesulfonic acid, the molecular weights were found to be 17,000. Their amino acid sequences were determined by alignment of peptides obtained by lysyl endopeptidase digestion and Staphylococcus aureus V8 protease digestion. PLI-A and PLI-B were each composed of 147 amino acid residues with one residue, Asn103, being for N-linked glycosylation, and the molecular weights of their protein portions were calculated to be 16,368 and 16,408, respectively. Each subunit contained four cysteine residues, all of which exist in disulfide linkages (Cys64-Cys141 and Cys119-Cys133). The sequences of PLI-A and PLI-B showed 89.9% homology to each other. When the sequences were compared with those of lipocortins, no significant homologies were detected. But the sequences were significantly homologous to those of COOH-terminal carbohydrate recognition portions of pulmonary surfactant apoprotein and animal lectins.

Peptide behaviour and analysis on a chemically stable C 18-bonded vinyl alcohol copolymer column with alkaline and acidic eluents

C 18-bonded vinyl alcohol copolymer (ODP) gel showed no weight loss or decrease in column efficiency for alkyl alcohols after being immersed in aqueous solutions of pH 2 and 10 at 50°C for 48 h, and only a 1% weight loss and a slight decrease in alkyl alcohol retention volumes after similar immersion at pH 13. The chemical stability of the ODP gel was further demonstrated in analyses of acidic, neutral and basic peptides on an ODP column with eluents of pH 3–10, which showed that the peptides differ considerably in the sensitivity of their retention behaviour to eluent pH, even though hydrophobic interaction invariably appeared to be the main retention mechanism. The ODP column was therefore applied to the analysis and alignment of lysil-endopeptidase (LEP) peptides derived from reduced and S-carboxymethylated carboxyl proteinase (Rcm-P-CP) of Pseudomonas sp. 101. One acid-soluble and seven alkaline-soluble LEP peaks were found in analyses on the ODP column using acidic and alkaline eluents, respectively. The chymotryptic peptides of Rcm-P-CP were first separated on an ODS column with an acidic eluent, and the eight elutes which contained lysine residue, as determined by amino acid analysis, were then analysed on the ODP column with an alkaline eluent, resulting in a further separation of each into several peaks and thus in the recovery of fractions of pure peptides. The LEP peptide alignment was then determined by overlapping the sequences of the chymotryptic peptides with the C- and N-terminal regions of the LEP peptides.

Carnivora: The Primary Structure of Hemoglobin from the Masked Palm Civet (Paguma larvata,Viverridae)

The primary structure of the alpha- and beta-chains of hemoglobin from the Masked Palm Civet (Paguma larvata, Viverridae) is described. The chains were separated directly from hemoglobin by RP-HPLC. After tryptic digestion of the chains, the peptides were separated by RP-HPLC. Amino acid sequences were determined by Edman degradation in liquid and gas-phase sequencers. The alignment of the tryptic peptides was made by homology with human and other Carnivora hemoglobins. Paguma and human hemoglobin differ with respect to 23 amino-acid residues. Some of these amino-acid substitutions, which occur in both the alpha- and beta-chains, occur at contact sites between the subunits, and at the binding sites of heme and of organic phosphate, as well as involving residues responsible for the alkaline Bohr effect.

Structure of bovine milk lipoprotein lipase

The primary structure of bovine milk lipoprotein lipase (bLPL) was determined by alignment of peptides produced by tryptic digestion, Staphylococcus aureus V8 protease digestion, and cyanogen bromide cleavage. bLPL consists of 450 amino acid residues. Most tryptic peptides were isolated and analyzed, except for the dipeptide, Glu-Lys (position 423-424), and the 2 Lys at positions 416 and 488. Peptides resulting from digestion by S. aureus V8 protease and cyanogen bromide cleavage filled the missing part and completed the primary sequence of bLPL. The NH2 terminus of bLPL was determined to be Asp by sequencing the intact protein with a gas phase sequencer for up to 30 residues, whereas the COOH terminus was identified as Gly through, carboxyl peptidase Y cleavage. The enzyme contains 10 cysteine residues, all of which exist in disulfide linkages. They are formed between Cys29 and Cys42, Cys218 and Cys241, Cys266 and Cys285, Cys277, and Cys280, and Cys420 and Cys440. The sites of N-glycosylation were identified at Asn44 and Asn361. In accordance with a common structural homology of serine-type esterases, -G-X-S-X-G- (Yang, C. Y., Manoogian, D., Pao, Q., Lee, F., Knapp, R. D., Gotto, A. M., Jr., and Pownall, H. J. (1987) J. Biol. Chem., 262, 3086-3191), the active site serine of bLPL was assigned to the serine at position 134. The chymotrypsin nick of bLPL was determined to be between residues 390 and 391. A model of the enzyme is proposed on the basis of our data and available chemical data.

Primary structure of the cytoplasmic domain of human erythrocyte protein band 3. Comparison with its sequence in the mouse.

We report here the peptide profile of the human cytoplasmic domain of band 3 protein (CDB-3). The peptide alignment was designed allowing for maximal homology with the murine protein whose sequence was deduced from cDNA analysis by Kopito and Lodish (Kopito, R.R., Anderson, M. and Lodish, H.F. (1987) J. Biol. Chem. 262, 8035-8040). In the human protein, part of the amino acid sequence has been determined by Kaul et al. (Kaul, R.K., Murthy, P.S.N., Reddy, A.G., Steck. T.L. and Kohler, H. (1983) J. Biol. Chem. 258, 7981-7990). We have sequenced most of the fragment not described by these author. The homology with the murine protein is high (90%), except in a few peptides where it is only 50%. The actual miniaturization of the techniques allows for the determination of a clear peptide profile of human CDB-3 starting from 10 ml blood samples. Our characterization of the peptide profile of membrane proteins is the first step towards the identification of genetic mutations, which have to be looked for in hemolytic anemia when the presence of an abnormal membrane protein is suspected.

The Hemoglobins of the Adult Blackbird(Turdus merula,Passeriformes). The Sequence of the Major (HbA) and Minor Component (HbD)

The blood of the adult blackbird contains one major hemoglobin component (HbA = alpha A2, beta 2, ca. 80%) and one minor one (HbD = alpha D2 beta 2, ca. 20%). The Hb-components were separated by FPLC on a TSK SP-5 PW column, and eluted with a linear NaCl gradient, while the globin chains were purified on a cation exchange (CM-Cellulose). Tryptic peptides from the globin chains were separated by HPLC on an RP-2 Lichrosorb column. The complete amino acid sequence was determined by automatic Edman degradation, using film and gas phase methods. For the alpha A-, alpha D- and beta-chains, peptide alignment was carried out relative to the corresponding chains of the greylag goose (Anser anser). The close phylogenetic relationship between blackbird, tree sparrow and starling is verified by the hemoglobin sequence. The O2-affinities of the major and minor hemoglobin components of the blackbird are not yet known. Thus, the results were interpreted on the basis of primary structure. Substitutions of possible structural significance were examined with the help of molecular graphics/modelling.

The amino acid sequence of Indian peafowl (Pavo cristatus) lysozyme and its comparison with lysozymes from phasianoid birds.

The amino acid sequence of Indian peafowl egg-white lysozyme has been identified. The reduced and carboxy methylated lysozyme was digested with trypsin followed by purification of the resulting peptides by reverse-phase HPLC. The tryptic peptides obtained were sequenced using the DABITC/PITC double coupling manual sequencing method. The alignment of the tryptic peptides were deduced by comparison with corresponding peptides of hen egg-white lysozyme. This protein proved to consist of 129 amino acid residues, and a relative molecular mass of 14423 Da was calculated. Amino acid sequence comparison of peafowl lysozyme and other phasianoid bird lysozymes revealed a maximum homology ratio of 98% with turkey lysozyme.

The amino acid sequence of barley rootlet trypsin inhibitor.

The amino acid sequence of barley rootlet trypsin inhibitor (BRTI) has been established. The reduced and S-carboxymethylated BRTI was cleaved with cyanogen bromide (CNBr), and three fragments (I, II, and III) were separated using gel filtration on a Bio-Gel P-30 column. Each CNBr-fragment was digested with trypsin and the resulting tryptic peptides were sequenced either by manual Edman degradation or by the DABITC/PITC double-coupling method. Alignment of the tryptic peptides from CNBr-fragment CB-I, which is the largest fragment, was done by analyzing the chymotrypic and thermolytic peptides derived from the CNBr-fragment CB-I. Thus, the amino acid sequence of BRTI consisting of 124 amino acid residues was established. BRTI proved to have intramolecular homology of 55% between the N-terminal half (positions 1 ~62) and the C-terminal half (positions 63 ~ 124); it also displays intermolecular homology with the trypsin inhibitors from wheat germ, rice bran, and soybeans. By comparing with soybean Bowman-Bir...

A domain of synapsin I involved with actin bundling shares immunologic cross-reactivity with villin.

Synapsin I is a neuronal phosphoprotein that can bundle actin filaments in vitro. This activity is under phosphorylation control, and may be related to its putative in vivo role of regulating the clustering and release of small synaptic vesicles. We have compared human and bovine synapsin I by peptide mapping, and have used NTCB (2-nitro-5-thiocyano benzoic acid) cleavage to generate a series of peptide fragments from bovine synapsin I. After chymotryptic digestion, 88% of the tyrosine-containing fragments appear to be structurally identical in human and bovine synapsin I, as judged by their positions on high-resolution two-dimensional peptide maps. The alignment of the NTCB peptides within the parent protein have been determined by peptide mapping, and the ability of these fragments to precipitate with actin bundles has been measured. Only peptides that are derived from regions near the ends of the protein are active. One such 25-kDa peptide which sediments with actin also cross-reacts with antibodies to chicken villin, an actin binding and bundling protein derived from the intestinal microvillus. Since in other respects villin appears to be an unrelated protein, these results suggest the possibility that certain actin binding proteins may show immunologic cross-reactivity due to convergent evolution within the acting binding domain.

Lecithin:cholesterol acyltransferase. Functional regions and a structural model of the enzyme.

The amino acid sequence of human lecithin:cholesterol acyltransferase has been determined by degradation and alignment of peptides obtained from tryptic and staphylococcal digestions and the cleavage with cyanogen bromide and consisted of 416 amino acid residues. All of the tryptic peptides of lecithin:cholesterol acyltransferase were isolated and sequenced. Peptides resulting from digestion by staphylococcal protease, cyanogen bromide cleavage, or the combination of the two methods were employed to find overlapping segments. The N terminus of human lecithin:cholesterol acyltransferase was determined to be phenylalanine by sequencing the whole protein up to 40 residues while the C terminus was identified as glutamic acid through carboxypeptidase Y cleavage. Cys50 and Cys74 and Cys313 and Cys356 were identified as the two disulfide bridges while the free sulfhydryl groups were located at positions 31 and 184. The N-glycosylated sites of the protein were assigned to asparagines at positions 20, 84, 272, and 384. The active site of lecithin:cholesterol acyltransferase was identified as serine on position 181 according to its homology with other serine-type esterases which have a common structure of glycine-variable amino acid-active serine-variable amino acid-glycine (Gly-X-Ser-X-Gly) with the variable amino acids disrupting the homology. No long internal repeats or homologies with apolipoproteins were found. The secondary structure is consistent with the results of predictive algorithms. A simple model of the enzyme is proposed on the basis of available chemical data and predictive methods.