Isolation,identification and synthesis of locustapyrokinin II from Locusta migratoria,another member of the FXPRL-amide peptide family

1. A blocked decapeptide was isolated from brain corpora cardiaca-corpora allata sub-oesophageal ganglion extracts of the locust, Locusta migratoria. Biological activity was monitored during HPLC purification by observing the myotropic effect of column fractions on the isolated hindgut of Leucophaea maderae.2. The primary structure of this myotropic peptide was established as: pGlu-Ser-Val-Pro-Thr-Phe-Thr-Pro-Arg-Leu-NH₂.3. The Chromatographic and biological properties of the synthetic peptide were the same as those of the native peptide, thus confirming structural analysis.4. This decapeptide is the sixth natural analog of a series of locust peptides with a Phe-X-Pro-Arg-Leu-NH₂ carboxyterminus. This carboxyl terminal sequence is also found in other peptides identified in other insects and it is the biological active core sequence for diverse biological activities: muscle contraction, pheromone production, pigment synthesis and diapauze.5. Like the locustamyotropins and locustapyrokinin I, locustapyrokinin II stimulates contractions of the oviduct in Locusta.     

Primary structure of the S-peptide formed by digestion of rabbit liver fructose 1,6-biphosphatase with subtilisin.

Digestion of rabbit liver fructose 1,6-bisphosphatase with subtilisin followed by denaturation of the protein yields a peptide containing 60 amino acid residues, including the blocked NH₂-terminus. This peptide has the following sequence: Ac-Ala-Asp-Lys-Ala-Pr o-Phe-Asp-Thr-Asp-Ile-Ser-Thr-Met-Thr-Arg-Phe-Val-Met-Glu-Glu-Gly-Arg-Ly s-Ala-Gly-Gly-Thr-Gly-Glu-Met-Thr-Gln-Leu-Leu-Asn-Ser-Leu-Cys-Thr-Ala-Va l-Lys-Ala-Ile-Ser-Thr-Ala-Val-Arg-Lys-Ala-Gly-Ile-Ala-His-Leu-Tyr-Gly-Ile-Ala.     

Inhibition of chloroplast adenosine triphosphatase activity by adenosine triphosphatase inhibitor from beef heart mitochondria.

A new amino-acid sequence is proposed for silk fibroin peptide Cp, after automatic Edman degradation studies. The proposed sequence is: Gly-Ala-Gly-Ala-Gly-Ser-Gly-Ala-Ala-Gly-(Ser-Gly-(Ala-Gly)_n)₈ Tyr, where n is usually 2.     

Chicken smooth muscle myosin light chain kinase is acetylated on its NH2-terminal methionine

The reported cDNA structrre, of chicken smooth muscle myosin light chain kinase (smMLCK) encodes a protein of 972 residues (Olsonet al. Proc. Natl. Acad. Sci USA, 87: 2284–2288, 1990). The calculated Mr is 107, 534 whereas the estimate by SDS-PAGE is approximately 130, 000. Gibson and Higgins (DNA Sequence (in press)) have recently reported the possibility of errors, in the cDNA sequence for non-muscle MLCK and that the NH₂-terminus of both it and smMLCK may extend beyond the reported coding region. The native smMLCK is NH₂-terminally blocked. A CNBr peptide derived from smMLCK contains the NH₂-terminal sequence Asp-Phe-Arg-Ala corresponding to residues 2 to 4 in the smMLCK sequence indicating, that Met-1 is present. Using a limited thermolysin digest we isolated an NH₂-terminally blocked peptide by reversed-phase HPLC. This thermolytic peptide had a mass of approximately 797 by time of flight mass spectrometry. Amino acid analysis and Edman sequencing of a CNBr-subfragment of the thermolytic peptide indicated that it had the composition and sequence, (Met)-Asp-Phe-Arg-Ala-Asn, with a calculated mass of 753. The difference in mass corresponds to the NH₂-terminal Met being blocked by actylation. The results demonstrate that the NH₂-terminal sequence of smMLCK inferred from the reported cDNA sequence is correct and that the proposed initiating, Met is not removed, but modified by -NH₂ acetylation of the translation product.     

Manipulation of peptide conformations by fine-tuning of the environment and/or the primary sequence

The widely observed phenomenon that peptides are capable of adopting multiple conformations in different environments suggests that secondary structure formation in a peptide segment is a process involving not only the peptide itself hut also the surrounding solvent media. The influence of the primary sequence and the molecular environment on peptide conformations are now investigated using synthetic peptides of amino acid sequence H₂N-(Ser-Lys)₂-Ala-X-Gly-Ala-X-Gly-Trp-Ala-X-Gly-(Lys-Ser)₃-OH, where X = Ile or Val. These two peptides, namely 3I (X = Ile) and 3V (X = Val), are found to lack defined secondary structure in aqueous buffer. However, discrete conformational states, e.g., α-helices and β-sheets, are readily generated and interconverted for both peptides when the buffer is modulated with the addition of methanol, sodium dodecyl sulfate (SDS) micelles, or phospholipid vesicles. The role of the primary sequence in affecting peptide conformations is manifested in that peptides 3I and 3V, which differ respectively in their content of β-branched Ile or Val residues, differ in their secondary structures at monomeric concentrations in 2 mM SDS and in mixed lipid vesicles of phosphatidic acid and phosphatidylcholine. The overall results suggest that peptide segments can be conformationally flexible entities poised to react to minor modulations in cither the molecular environment or the primary sequence, a circumstance that may he relevant to protein functioning and folding. © 1995 John Wiley & Sons, Inc.     

A peptide study of the relationship between the collagen triple-helix and amyloid

Type XXV collagen, or collagen‐like amyloidogenic component, is a component of amyloid plaques, and recent studies suggest this collagen affects amyloid fibril elongation and has a genetic association with Alzheimer's disease. The relationship between the collagen triple helix and amyloid fibrils was investigated by studying peptide models, including a very stable triple helical peptide (Pro‐Hyp‐Gly)₁₀, an amyloidogenic peptide GNNQQNY, and a hybrid peptide where the GNNQQNY sequence was incorporated between (GPO)_n domains. Circular dichroism and nuclear magnetic resonance (NMR) spectroscopy showed the GNNQQNY peptide formed a random coil structure, whereas the hybrid peptide contained a central disordered GNNQQNY region transitioning to triple‐helical ends. Light scattering confirmed the GNNQQNY peptide had a high propensity to form amyloid fibrils, whereas amyloidogenesis was delayed in the hybrid peptide. NMR data suggested the triple‐helix constraints on the GNNQQNY sequence within the hybrid peptide may disfavor the conformational change necessary for aggregation. Independent addition of a triple‐helical peptide to the GNNQQNY peptide under aggregating conditions delayed nucleation and amyloid fibril growth. The inhibition of amyloid nucleation depended on the Gly‐Xaa‐Yaa sequence and required the triple‐helix conformation. The inhibitory effect of the collagen triple‐helix on an amyloidogenic sequence, when in the same molecule or when added separately, suggests Type XXV collagen, and possibly other collagens, may play a role in regulating amyloid fibril formation. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 795–806, 2012.     

Location and primary structure of the substrate binding site peptide of aldolase C

An octadecapeptide containing the substrate-combining site of rabbit brain aldolase (aldolase C) has been isolated. This peptide has tentatively been assigned the structure: Ala-Leu-Ser(Asx,His,His,Val,Tyr)(Leu,Glx,Gly,Thr,Leu,Leu)(Lys,Pro,Asx,Met). The primary sequence of this peptide thus appears to be very similar to that of the active-site peptide of rabbit muscle aldolase (aldolase A), but it is located in a different BrCN segment, approximately 50 residues closer to the NH₂-terminus of the aldolase C subunit. A tentative sequence has also been obtained for an adjacent nonapeptide, also homologous with the corresponding structure in aldolase A. The evidence suggests that a large segment of the peptide chain in aldolase C may be translocated, as compared with aldolase A.     

Sequence specificity of the PHSRN peptide from fibronectin on corneal epithelial migration

The Pro-His-Ser-Arg-Asn (PHSRN) sequence in fibronectin is a second cell-binding site that synergistically affects with Arg-Gly-Asp. The PHSRN peptide also induces cell invasion and accelerates wound healing. Here, we examined the sequence specificity of PHSRN on corneal epithelial migration using various synthetic peptides. Elongation and deletion analyses of Ac-PHSRN-NH₂ suggest that the five amino acid length was a minimum and essential sequence for promotion of rabbit corneal epithelial migration ex vivo. Additionally, alanine substituted analysis indicated that the Ser- and Arg-residues are critical for the biological activities. The Ser-Arg motif is involved in various biologically active peptides, suggesting that the unique sequence interacts with cellular receptor(s) and regulates biological functions. Further, the N-acetyl and C-amide of Ac-PHSRN-NH₂ contributed effectively for the chemical stability in tears. The Ac-PHSRN-NH₂ peptide has potential to use as a therapeutic reagent especially for corneal wound healing.     

Increasing the activity of cell adherent cyclic NGR peptides by optimizing the peptide length and amino acid character

Cyclic peptides are an attractive modality for the development of therapeutics and the identification of functional cyclic peptides that contribute to novel drug development. The peptide array is one of the optimization methods for peptide sequences and also useful to understand sequence–function relationship of peptides. Cell adherent cyclic NGR peptide which selectively binds to the aminopeptidase N (APN or CD13) is known as an attractive tumor marker. In this study, we designed and screened a library of different length and an amino acid substitution library to identify stronger cell adhesion peptides and to reveal that the factor of higher binding between CD13 and optimized cyclic peptides. Additionally, we designed and evaluated 192 peptide libraries using eight representative amino acids to reduce the size of the library. Through these optimization steps of cyclic peptides, we identified 23 peptides that showed significantly higher cell adhesion activity than cKCNGRC, which was previously reported as a cell adhesion cyclic peptide. Among them, cCRHNGRARC showed the highest activity, that is, 1.65 times higher activity than cKCNGRC. An analysis of sequence and functional data showed that the rules which show higher cell adhesion activity for the three basic cyclic peptides (cCX₁HNGRHX₂C, cCX₁HNGRAX₂C, and cCX₁ANGRHX₂C) are related with the position of His residues and cationic amino acids.     

Modification of yeast 3-phosphoglycerate kinase: isolation and sequence determination of a nitrated active-site peptide and isolation of a carboxyl modified active-site peptide.

Previous studies have shown that yeast 3-phosphoglycerate kinase is inhibited by nitration of a single tyrosine residue. Chymotryptic fragmentation of the nitrated protein followed by peptide mapping revealed approximately fifty peptides, one of which was shown to contain a nitrotyrosine residue. Isolation of this unique peptide was accomplished by gel filtration and high voltage paper electrophoresis. The sequence as established by Edman degradation and carboxypeptidase hydrolysis is: Lys-NO₂Tyr-Phe-Phe-Lys. Independent observations on the X-ray crystallographic model of yeast phosphoglycerate kinase provides supportive evindence of this sequence. Additionally, a peptide has been isolated containing an active-site carboxyl residue following modification of the enzyme with [¹⁴C]methoxyamine.     

Multi-modal Binding of a ‘Self’ Peptide by HLA-B*27:04 and B*27:05 Allelic Variants,but not B*27:09 or B*27:06 Variants: Fresh Support for Some Theories Explaining Differential Disease Association

A self-derived-peptide with the same amino acid sequence (N-RRYLENGKETLQR-C) as residues 169–181 of the human leukocyte antigen (HLA) B27 heavy chain is known to bind to MHC Class I complexes containing the HLA-B27 heavy chain. This observation has been invoked previously in at least two different (but related) molecular explanations for the disease-association of the HLA-B27 allele. Here, we use a combination of fluorescence polarization, competitive inhibition and gel filtration chromatographic studies to show that a fluorescently-labeled peptide of the above sequence binds to two disease-associated subtypes of HLA-B27 (namely HLA-B*27:04 and HLA-B*27:05) but not to non-disease-associated subtypes (HLA-B*27:06 or HLA-B*27:09). This differential binding behavior is seen both in (a) peptide binding to complexes of heavy chain (HLA-B27) and light chain (β₂ microglobulin), and in (b) peptide binding to β₂ microglobulin-free heavy chains in the aggregated state. Such subtype-specific differences are not seen with two other control peptides known to bind to HLA-B27. Our results support the likelihood of differential peptide binding holding at least one of the keys to HLA-B27’s disease association.     

An approach to peptide-based ATP receptors by a combination of random selection, rational design, and molecular imprinting

Random selection, rational design and molecular imprinting were cooperatively utilized to develop peptide-based ATP synthetic receptors. In this fusion strategy, combinatorial chemistry was utilized for screening a precursor peptide useful for construction of ATP receptors, and rational design was employed in modification of the selected precursor peptide for higher affinity and selectivity. Finally, molecular imprinting was used for pre-organizing the conformation of the precursor peptide as complementary to a target molecule ATP. The fusion strategy appeared to have advantage to sole use of the individual strategy: (1) a low hit-rate of combinatorial chemistry will be improved by customizing a higher order structure of a selected peptide by molecular imprinting, (2) combinatorial chemistry allows us to semi-automatically select components of water-compatible synthetic receptors, (3) rational design improves the selected peptide sequence for better molecularly imprinted receptors.A peptide consisting of a randomly selected sequence and a rationally designed sequence (Resin-Lys-Gly-Arg-Gly-Lys-Gly-Gly-Gly-Glu-Lys-Tyr-Leu-Lys-NHAc) was designed and synthesized as a precursor peptide. The rational design was made according to the sequence of the adenine binding site of biotin carboxylase. The on-beads peptide was cross-linked with dimethyl adipimidate in the presence of ATP. In the saturation binding tests, the cross-linked on-beads peptide showed 5.3 times higher affinity compared to the non-cross-linked peptide with the same sequence. Furthermore, the cross-linked peptide showed improved selectivity; the ratios of binding constants, K_(ATP)/K_(ADP) and K_(ATP)/K_(GTP), were increased from 2.4 to 19, and from 0.8 to 10, respectively. It would be notable that the peptide without the rationally designed sequence showed no discrimination between ATP and GTP (K_(ATP)/K_(GTP) as 0.9), suggesting that the rationally designed site was successfully engaged for recognition of the adenine base.     

Identification and synthesis of a heptapeptide in uremic fluid

A heptapeptide isolated from uremic fluid was synthesized by conventional method. The total amino acid sequence of this peptide was deduced as follows: H-His-Pro-Ala-Glu-Asn-Gly-Lys-OH. Structural similarity was soon realized between this peptide and heptapeptde moiety corresponding to position 13 through 19 of β₂-microglobulin.     

Studies on the structure of rabbit muscle aldolase: Amino acid sequence of cysteine-containing peptides

Five cysteine-containing peptides have been isolated in nearly stoichemometric yields from the tryptic digests of the NH₂^? and COOH-terminal BrCN peptides of rabhit muscle aldolase and their sequence determined. Peptides NS1, NS2, and NS3, from the NH₂-terminal part of the enzyme have the following sequences: NS1, Val-Asp-Pro-Cys-Ile-Gly-Gly-Val-Ile-Leu-Phe-His-Glu-Thr-Leu-Tyr-Gln-Lys; NS2, Cys-Val-Leu-Lys; NS3, Cys-Ala-Glu-Tyr-Lys. The two peptides isolated from the COOH-terminal region are: CS1, Ala-Leu-Ala-Asn-Ser-Leu-Ala-Cys-Gln-Gly-Lys and CS2, Cys-Pro-Leu-Leu-Trp-Pro-Lys-Ala-Leu-Thr-Phe-Ser-Tyr-Gly-Arg. The Lys-Ala bond in peptide CS2 was found to be resistant to tryptic hydrolysis. The results provide the basis for assigning the positions of cysteine residues in the polypeptide chain. Cys-72 in peptide NS1 and Cys-336 in peptide CS1 are the residues that form a disulfide bridge when the enzyme is inactivated by oxidation with an o-phenanthroline-Cu²⁺ complex; Cys-287 in peptide CS2 in one of the two exposed residues, while Cys-134 and Cys-149 in peptides NS2 and NS3, respectively, are buried in the native enzyme. All of eight cysteine-containing peptides of rabbit muscle aldolase have now been sequenced, and structural homology of the α and β subunits extended to these regions.     

Isolation and primary structure of neuropeptides from the mosquito,Aedes aegypti,immunoreactive to FMRFamide antiserum

Two novel neuropeptides, Aea-HP-I and II, have been isolated from a head extract of the mosquito, Aedes aegypti; they were detected by a FMRFamide radioimmunoassay. The peptides were purified by gel filtration, ion exchange chromatography, and reversed-phase high performance liquid chromatography. Amino acid composition and sequence analysis, combined with enzymatic digestion, established the primary structure of Aea-HP-I as pGlu-Arg-Pro-Hyp-Ser-Leu-Lys-Thr-Arg-Phe-NH₂ and Aea-HP-II as Thr-Arg-Phe-NH₂. Aea-HP-I was synthesized, and chromatographic properties of the synthetic peptide were the same as those of the native peptide, thus confirming the structural analysis. The peptide has three unusual residues: an amino-terminal pGlu, a Hyp in the fourth position, and a carboxyl-terminal amide. The Pro-Hyp sequence occurs in toxin peptides from the venoms of cone snails and wasps and in bradykinin analogues. Although the functions of Aea-HP-I and II have not been determined, the peptides have the same RFa sequence at the carboxyl-terminal as Lem-SK-I and II (leucosulfakinins) and Lem-MS (leucomyosuppressin) in cockroaches and FMRFamide-related peptides in molluscs.     

Partial Characterization of a Novel Cardioinhibitory Peptide from the Brain of the Snail Helix aspersa

1. We report the isolation of a peptide from the brain of the snail Helix aspersa by radioimmunoassay using an antisomatostatin.2. The sequencing of an immunopositive fraction showed the presence of a new tridecapeptide, termed Helix cardioinhibitory peptide (HCIP), with the following primary structure : H-Val-Phe-Gln-Asn-Gln-Phe-Lys-Gly-Ile-Gln-Gly-Arg-Phe-NH₂. It is structurally related to the Achatina cardioexcitatory peptide (ACEP-1) and the terminal-ammo acid sequence of HCIP is identical to that of FMRFamide family peptides.3. The synthetic HCIP was tested on heart and neuronal activities and it was found to have inhibitory actions not only on the ventricle but also on visceral neurons of the central nervous system of Helix. Immunocytochemical investigation indicates its presence in visceral and parietal ganglia, in which cells taking part in the regulation of the heartbeat have been previously identified .     

Theoretical structure of the polar regions of the tropocollagen molecule

The theoretical conformations of poly (Gly-Ala-Glu) have been studied. This peptide was chosen as a model for the glycine led triads of the polar regions in collagen. The most favorable conformations are found to be based on the extended and folded forms of the 2₇ helix (2_7a and 2_7b). It is suggested that triple-strand structures of folded 2₇ helices exist in the polar collagen regions, and a structural model is presented which is in accord with recent ultrastructural deformation studies. It is a necessary condition for this structure that glycine occur in the lead of the peptide triads. In regions of the collagen molecule where the primary sequence does not contain triads (e.g., in the telopeptide region), random structures based on energy minimization of peptide neighbors are considered briefly. It seems likely that such regions contain an admixture of left-hand α, polyproline II, and 2₇ helix structures.     

Comparative peptide mapping at the nanomole level

Comparison of highly similar proteins by peptide mapping at the 1-nmol level on cellulose-coated thin-layer sheets is described in detail. Maps were developed in three steps (a) a short chromatographic run in butanol:pyridine:acetic acid:H₂O (150:100:3:100), (b) electrophoresis at pH 3.5, and (c) rechromatography in butanol:pyridine:acetic acid:H₂O. The inclusion of step (a) greatly reduced streaking in the subsequent electrophoretic direction. Peptides were located by fluorescamine staining and could be eluted for composition or sequence analysis. To ensure complete comparison of the proteins two types of proteolytic digestion, a tryptic and a combined tryptic/thermolytic digestion, were necessary.     

Crystal structure of the collagen model peptide (Pro‐Pro‐Gly)4‐Hyp‐Asp‐Gly‐(Pro‐Pro‐Gly)4 at 1.0 Å resolution

The single‐crystal structure of the collagen‐like peptide (Pro‐Pro‐Gly)₄‐Hyp‐Asp‐Gly‐(Pro‐Pro‐Gly)₄, was analyzed at 1.02 Å resolution. The overall average helical twist (θ = 49.6°) suggests that this peptide adopts a 7/2 triple‐helical structure and that its conformation is very similar to that of (Gly‐Pro‐Hyp)₉, which has the typical repeating sequence in collagen. High‐resolution studies on other collagen‐like peptides have shown that imino acid‐rich sequences preferentially adopt a 7/2 triple‐helical structure (θ = 51.4°), whereas imino acid‐lean sequences adopt relaxed conformations (θ < 51.4°). The guest Gly‐Hyp‐Asp sequence in the present peptide, however, has a large helical twist (θ = 61.1°), whereas that of the host Pro‐Pro‐Gly sequence is small (θ = 46.7°), indicating that the relationship between the helical conformation and the amino acid sequence of such peptides is complex. In the present structure, a strong intermolecular hydrogen bond between two Asp residues on the A and B strands might induce the large helical twist of the guest sequence; this is compensated by a reduced helical twist in the host, so that an overall 7/2‐helical symmetry is maintained. The Asp residue in the C strand might interact electrostatically with the N‐terminus of an adjacent molecule, causing axial displacement, reminiscent of the D‐staggered structure in fibrous collagens. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 436–447, 2013.     

Enriching C-terminal peptide from endopeptidase ArgC digest for protein C-terminal analysis

This Letter describes a method for enriching C-terminal peptide of protein for C-terminal sequence analysis. This method employs endopeptidase ArgC digestion and C-terminal peptide enrichment using m-aminophenylboronic acid–agarose as an arginine-capture material. The selectively recovered C-terminal peptide incorporates no artificial derivatization. Therefore, the widely used functional groups (e.g. α-NH₂ and α-COOH) can be used for any necessary transformation. In this research, a TMPP mass tag was attached to the α-NH₂ group to clarify the amino acid sequence of the C-terminal peptide.