Two-level QSAR network (2L-QSAR) for peptide inhibitor design based on amino acid properties and sequence positions

In the design of peptide inhibitors the huge possible variety of the peptide sequences is of high concern. In collaboration with the fast accumulation of the peptide experimental data and database, a statistical method is suggested for peptide inhibitor design. In the two-level peptide prediction network (2L-QSAR) one level is the physicochemical properties of amino acids and the other level is the peptide sequence position. The activity contributions of amino acids are the functions of physicochemical properties and the sequence positions. In the prediction equation two weight coefficient sets {a_k} and {b_l} are assigned to the physicochemical properties and to the sequence positions, respectively. After the two coefficient sets are optimized based on the experimental data of known peptide inhibitors using the iterative double least square (IDLS) procedure, the coefficients are used to evaluate the bioactivities of new designed peptide inhibitors. The two-level prediction network can be applied to the peptide inhibitor design that may aim for different target proteins, or different positions of a protein. A notable advantage of the two-level statistical algorithm is that there is no need for host protein structural information. It may also provide useful insight into the amino acid properties and the roles of sequence positions.     

多级质谱法测定Axinastatin 1环肽序列及其断裂机理研究

对具有抗癌活性的海洋环肽Axinastatin 1进行化学合成. 采用多级质谱法对合成环肽进行序列测定. 线性前体测序依据b_x－y_z断裂路径, 在同一张MS²谱中利用b和y离子所提供序列信息的互补来实现. 环肽测序依据b_x→b_x－1断裂路径, 每一级MS由b离子的C端碰撞掉一个氨基酸残基直到MS⁶, 得到2套b离子, 根据它们所提供序列信息的互补可准确测定环肽序列并推断其环结构, 同时观察到b离子重排现象. 讨论了上述断裂与重排的路径和机理, 并利用半经验量子化学PM3和AM1两种算法计算了碎片的生成焓, 验证了路径的合理性. 由离子b_5PN的生成焓偏高和其重排间的联系尝试提出过渡结构假设.     

Stapled Peptides with γ‐Methylated Hydrocarbon Chains for the Estrogen Receptor/Coactivator Interaction

“Stapled” peptides are typically designed to replace two non‐interacting residues with a constraining, olefinic staple. To mimic interacting leucine and isoleucine residues, we have created new amino acids that incorporate a methyl group in the γ‐position of the stapling amino acid S5. We have incorporated them into a sequence derived from steroid receptor coactivator 2, which interacts with estrogen receptor α. The best peptide (IC₅₀=89 nm ) replaces isoleucine 689 with an S‐γ‐methyl stapled amino acid, and has significantly higher affinity than unsubstituted peptides (390 and 760 nm ). Through X‐ray crystallography and molecular dynamics studies, we show that the conformation taken up by the S‐γ‐methyl peptide minimizes the syn‐pentane interactions between the α‐ and γ‐methyl groups.     

Amino acid identification and sequence analysis of peptides by reaction mass spectrometry

Fast atom bombardment mass spectrometry (FAB-MS) is applied to distinguish N-terminal series ions from C-terminal series ions of a peptide by on-probe acetylation, it providesvaluable information about the sequence of an unknown peptide. The FAB mass spectra containa number of characteristic ions at low-mass region in addition to the sequence ions at high-massregion. It was found that the ions below m/z 200 are characteristic of the amino acid composition ofthe peptide, from which the amino acid composition of the peptide could be estimated. Additionally,mixture analysis is also discussed.     

Synthesis of the human adrenal cortex hormone ( h ACTH) with a revised amino acid sequence

We demonstrated recently that the amino acid sequence of human ACTH (α_h-ACTH¹) differs in four positions from the structure reported in 1961 by Lee and coworkers. The synthesis of the revised sequence and a method of verifying the identity of the synthetic peptide with the naturally occurring hormone are described. The two preparations can most readily be compared by examining the mixture of fragments obtained upon degradation with trypsin. In this mixture, fragments 22–39 of natural α_h-ACTH and of the synthetic peptide are identical and easily distinguishable from the corresponding synthetic fragment of the previously postulated sequence. This is demonstrated by thin layer-chromatography and -electrophoresis as well as by reference to the rate of deamidation under alkaline conditions.     

Cloning and Characterization of a Novel Aspartic Protease Gene from Marine-Derived <Emphasis Type="Italic">Metschnikowia reukaufii</Emphasis> and its Expression in <Emphasis Type="Italic">E. coli</Emphasis>

Metschnikowia reukaufii W6b isolated from marine environment was found to produce a cell-bound acid protease. The full-length cDNA (cDNASAP6 gene) of the acid protease (SAP6) from the marine-derived yeast M. reukaufii W6b was cloned. The insert was 1,755-bp long and contained an open reading frame of 1,527-bp encoding 508 amino acids. The deduced amino acid sequence included a signal peptide of 16 amino acids. The consensus motifs contained a VLLDTGSSDLRM active site and an ALLDSGTTITQF active site. The protein sequence deduced from the cDNASAP6 gene exhibited 12.9% overall identity with Cwp1 of Saccharomyces cerevisiae and a hydropathy profile characteristic of glycosylphosphatidylinositol cell-wall proteins. The cDNASAP6 gene without 48 bp encoding the signal peptide sequence was subcloned into an expression plasmid pET-24a (+) and fused with a 6-His Tag and transformed into Escherichia coli BL21 (DE3) for recombinant expression of the protease. The expressed fusion protein was found to have a unique band with molecular mass of about 54 kDa. The crude acid protease of the culture of the marine yeast strain W6b and the crude recombinant acid protease had milk clotting activity.     

Preparation of N‐Fmoc‐Protected (S)‐5‐Amino‐4,4‐difluoro‐7‐methyloctanoic Acid,a Possible Dipeptide Isostere

The title compound 1 was prepared from L ‐leucine. The key steps include a Grignard addition to Bn₂‐leucinal, a CO/CF₂ replacement with Et₂NSF₃ (DAST) and use of a Ph group as synthetic equivalent of a COOH group. The difluoro‐δ‐amino acid 1 was incorporated into a peptide 8 ; tests with various proteases showed no inhibition by this particular peptide.     

C-Glycoside Analogues of N4-(2-Acetamido-2-deoxy-β-D-glucopyranosyl)-L-asparagine: Synthesis and conformational analysis of a cyclic C-glycopeptide

The synthesis of C-glycosidic analogues 15–22 of N⁴-(2-acetamido-2-deoxy-β-D -glucopyranosyl)-L -asparagine (Asn(N⁴GlcNAc)) possessing a reversed amide bond as an isosteric replacement of the N-glycosidic linkage is presented. The peptide cyclo(-D -Pro-Phe-Ala-CGaa-Phe-Phe-) (CGaa = C-glycosylated amino acid; 24 ) was prepared to demonstrate that 3-[(3-acetamido-2,6-anhydro-4,5,7-tri-O-benzyl-3-deoxy-β-D -glycero-D -guloheptonoyl)amino]-2-[(9H-fluoren-9-yloxycarbonyl)amino]propanoic acid ( 22 ) can be used in solid-phase peptide synthesis. The conformation of 24 was determined by NMR and molecular-dynamics (MD) techniques. Evidence is provided that the CGaa side chain interacts with the peptide backbone. The different C-glycosylated amino acids 15–21 were prepared by coupling 3-acetamido-2,6-anhydro-4,5,7-tri-O-benzyl-3-deoxy-β-D -glycero-D -gulo-heptonic acid ( 4 ) with diamino-acid derivatives 8–14 in 83–96% yield. The synthesis of 4 was performed from 2-(acetamido-3,4,6-tri-O-benzyl-2-deoxy-β-D -glucopyranosyl) tributylstannane ( 2 ) by treatment with BuLi and CO₂ in 83% yield. Similarly, propyl isocyanat yielded the glycoheptonamide 7 in 52% from 2 . Compound 2 was obtained from 2-acetamido-3,4,6-tri-O-benzyl-2-deoxy-D -glucopyranose ( 1 ) by chlorination and addition of tributyltinlithium in 74% yield. A procedure for a multigram-scale synthesis of 1 is given.     

Factors affecting immonium ion intensities in the high-energy collision-induced decomposition spectra of peptides

Each amino acid in a peptide has a characteristic immonium ion (H₂N⁺?CHR), the presence of which in a mass spectrum can indicate the presence of that amino acid. High-energy collision-induced decomposition studies on small peptide ions formed by fast atom bombardment showed the relative intensities of these immonium ions to be dependent on the relative positions of the amino acids in the peptide chain: C-terminal, N-terminal or in-chain. Evidence in favour of competition in the formation of immonium ions is presented.     

Preferred 3D‐Structure of Peptides Rich in a Severely Conformationally Restricted Cyclopropane Analogue of Phenylalanine

Terminally blocked, homo‐peptide amides of (R,R)‐1‐amino‐2,3‐diphenylcyclopropane‐1‐carboxylic acid (c₃diPhe), a chiral member of the family of C^α‐tetrasubstituted α‐amino acids, from the dimer to the tetramer, and diastereomeric co‐oligopeptides of (R,R)‐ or (S,S)‐c₃diPhe with (S)‐alanine residues to the trimer level were prepared in solution and fully characterized. The synthetic effort was extended to terminally protected co‐oligopeptide esters to the hexamer, where c₃diPhe residues are combined with achiral α‐aminoisobutyric acid residues. The preferred conformations of the peptides were assessed in solution by FT‐IR absorption, NMR, and CD techniques, and for seven oligomers in the crystal state (by X‐ray diffraction) as well. This study clearly indicates that c₃diPhe, a sterically demanding cyclopropane analogue of phenylalanine, tends to fold peptides into β‐turn and 3₁₀‐helix conformations. However, when c₃diPhe is in combination with other chiral residues, the conformation preferred by the resulting peptides is also dictated by the chiral sequence of the amino acid building blocks. The (S,S)‐enantiomer of this α‐amino acid, unusually lacking asymmetry in the main chain, strongly favors the left‐handedness of the turn/helical peptides formed.     

Benzyl tert‐but­oxy­carbonyl‐α‐amino­isobutyrate

The small synthetic peptide, benzyl 2‐(tert‐but­oxy­carbonyl‐amino)­isobutyrate, C₁₆H₂₃NO₄, has the α‐helical conformation [|?| = 55.8 (2)° and |ψ| = 37.9 (2)°] observed in peptide fragments of peptaibols containing the α‐amino­isobutyric acid (Aib) residue. The structure shows no intramolecular hydrogen bonding, which would disrupt the limited conformational freedom associated with this amino acid. Two weak intermolecular hydrogen contacts are observed.     

A Selective HIV‐Protease Assay Based on a Chromogenic Amino Acid

(2S,3S)‐2‐Amino‐3‐hydroxy‐5‐(4‐nitrophenoxy)pentanoic acid ( 5 ) was prepared stereoselectively as the N‐Fmoc, O‐(tert‐butyl)‐protected derivative 5a in eleven steps from ethyl (E)‐4‐benzyloxypent‐2‐enoate ( 6 ). This protected amino acid was used for the solid‐phase peptide synthesis of oligopeptides, which serve as sequence‐specific chromogenic protease substrates when used in the presence of NaIO₄ and bovine serum albumin. The peptide 1 (KRAVNle 5  EANleNH₂ (Nle=norleucine)) allows detection of HIV‐protease activity spectrophotometrically at 405 nm.     

Thermodynamic Properties of Peptide Solutions. Part 18. Partial Molar Isentropic Compressibilities of Gly-X-Gly Tripeptides (X = Tyr,Pro, Gln,Asp and Glu), and the Peptide Salts K[GlyAspGly], Na[GlyGluGly] and GlyLysGly Acetate in Aqueous Solution at 25 <Superscript>∘</Superscript>C

The partial molar isentropic compressibilities at infinite dilution, K∘_S,2, have been determined for several tripeptides of the sequence glycyl-X-glycine, where X is one of the amino acids tyrosine, proline, glutamine, aspartic acid, glutamic acid and lysine in aqueous solution at 25 ^∘C. These results, along with those for triglycine, were used to estimate the contributions of the amino acid side-chains to the partial molar isentropic compressibilities of polypeptides. Values for K∘_S,2 have also been determined for aqueous solutions of the two peptide salts K[glyaspgly] and Na[glyglugly]. The K∘_S,2 results for the peptides and their salts have been combined with literature data for electrolytes to calculate the changes in isentropic compressibility upon ionization of the acidic side-chains. The results are compared with those for other carboxylic acid systems.     

N-terminal tagging strategy for De Novo sequencing of short peptides by ESI-MS/MS and MALDI-MS/MS

The major portion of skin secretory peptidome of the European Tree frog Hyla arborea consists of short peptides from tryptophyllin family. It is known that b-ions of these peptides undergo head-to-tail cyclization, forming a ring that can open, resulting in several linear forms. As a result, the spectrum contains multiple ion series, thus complicating de novo sequencing. This was observed in the Q-TOF spectrum of one of the tryptophyllins isolated from Hyla arborea; the sequence FLPFFP-NH₂ was established by Edman degradation and counter-synthesis. Though no rearrangements were observed in FTICR-MS and MALDI-TOF/TOF spectra, both of them were not suitable for mass-spectrometry sequencing due to the low sequence coverage. To obtain full amino acid sequence by mass spectrometry, three chemical modifications to N-terminal amino moiety were applied. They include acetylation and sulfobenzoylation of N-amino group and its transformation to 2,4,6-trimethylpyridinium by interaction with 2,4,6-trimethylpyrillium tetrafluoroborate. All three reagents block scrambling and provide spectra better than the intact peptide. Unfortunately, all of them also readily react with lysine side chain. Hence, all investigated procedures can be used to improve sequencing of short peptides, while acetylation is the recommended one. It shows excellent results, and it is plain and simple to perform. This is the procedure of choice for MS-sequencing of short peptides by manual or automatic algorithms.     

The role of the membrane-assisted hydrophobic interaction between di-, tri-, or tetrapeptide catalysts and amino acid esters for the enhancement of stereoselective hydrolysis reactions

The peptide catalyst having the amino acid sequence (Z-L -Leu (or Phe)-L -His) was found to be the most stereoselective among the L -histidyl group-containing di-, tri-, or tetrapeptide catalysts in the hydrolysis of enantiomeric amino acid substrates in vesicular membranes. The role of the membrane-assisted hydrophobic interaction between the peptide catalyst and enantiomeric substrates for the improvement of the hydrolysis stereoselectivity was demonstrated by means of direct measurements of nuclear Overhauser effect spectroscopy (NOESY) spectra of the interaction system and by the extremely high stereoselectivity itself obtained by intensifying the membrane-promoted hydrophobic interaction between the catalyst and the substrates.     

Proton affinities of the N- and C-terminal segments arising upon the dissociation of the amide bond in protonated peptides

Dissociation of the amide bonds in a protonated peptide leads to N-terminal sequence fragments with cyclic structures and C-terminal sequence fragments with linear structures. The ionic fragments containing the N-terminus (b _n ) have been shown to be protonated oxazolones, whereas those containing the C-terminus (y _n ) are protonated linear peptides. The coproduced neutral fragments are cyclic peptides from the N-terminus and linear peptides from the C-terminus. A likely determinant of these structural choices is the proton affinity (PA) of the described peptide segments. This study determines the PA values of such segments (Pep), i.e., cyclic and linear dipeptides and a relevant oxazolone, based on the dissociations of proton-bound dimers [Pep + B _i ]H⁺ in which B _i is a reference base of known PA value (Cooks kinetic method). The dissociations are assessed at different internal energies to thereby obtain both proton affinities as well as entropies of protonation. For species with comparable amino acid composition, the proton affinity (and gas phase basicity) follows the order cyclic peptide ≪ oxazolone ≈ linear peptide. This ranking is consistent with dissociation of the protonated peptide via interconverting proton-bound complexes involving N-terminal oxazolone (O) or cyclopeptide (C) segments and C-terminal linear peptide segments (L), viz. O ⋯ H⁺ ⋯ L ⇄ C ⋯ H⁺ ⋯ L. N-terminal sequence ions (b _n ) are formed with oxazolone structures which can efficiently compete for the proton with the linear segments. On the other hand, N-terminal neutral fragments detach as cyclic peptides, with H⁺ now being retained by the more basic linear segment from the C-terminus to yield y _n .     

Determination of glycosylation sites in O-linked glycopeptides: A sensitive mass spectrometric protocol

A sensitive mass spectrometric method for the specific identification of O-glycosylation sites in glycopeptides is presented. Reductive β-elimination liberates a peptide containing a specifically modified amino acid at the former site of carbohydrate attachment. Mass spectrometry is used to determine the sequence of the modified peptide, in which the position of the modified amino acid marks the site of O-glycosylation. The method also provides information on the carbohydrate chain and is applicable at the low nanomolar level.     

Gene Cloning,Expression, and Characterization of a Novel Phytase from <Emphasis Type="Italic">Dickeya paradisiaca</Emphasis>

A novel phytase gene, appA, was isolated by degenerate polymerase chain reaction (PCR) and thermal asymmetric interlaced PCR from Dickeya paradisiaca. The full-length appA comprises 1278 bp and encodes 425 amino acid residues, including a 23-residue putative N-terminal signal peptide. The deduced amino acid sequence of appA reveals the conserved motifs RHGXRXP and HD, which are typical of histidine acid phosphatases; significantly, APPA shows maximum identity (49%) to a phytase from Klebsiella pneumoniae. To characterize the properties of APPA, appA was expressed in Escherichia coli and purified. The purified recombinant APPA has two pH optima at pH 4.5 and 5.5, optimum temperature at 55 °C, specific activity of 769 U/mg, and good pH stability. The K _m value for the substrate sodium phytate is 0.399 mM with a V _max of 666 U/mg. To our knowledge, this is the first report of a phytase or phytase gene isolated from Dickeya. Weina Gu and Huoqing Huang contributed equally to this work.     

Radical-driven dissociation of odd-electron peptide radical ions produced in 157 nm photodissociation

Odd-electron a+1 radical ions generated in the 157 nm photodissociation of peptide ions were investigated in an ion trap mass spectrometer. To localize the radical, peptide backbone amide hydrogens were replaced with deuterium. When the resulting radical ions underwent hydrogen elimination, no H/D scrambling was obvious, suggesting that without collisional activation, the radical resides on the terminal α-carbon. Upon collisional excitation, odd-electron radical ions dissociate through two favored pathways: the production of a-type ions at aromatic amino acids via homolytic cleavage of backbone C_α-C(O) bonds and side-chain losses at nonaromatic amino acids. When aromatic residues are not present, nonaromatic residues can also lead to a-type ions. In addition to a-type ions, serine and threonine yield c _n−1 and a _n−1+1 ions where n denotes the position of the serine or threonine. All of these fragments appear to be directed by the radical and they strongly depend on the amino acid side-chain structure. In addition, thermal fragments are also occasionally observed following cleavage of labile Xxx-Pro bonds and their formation appears to be kinetically competitive with radical migration.     

Does in-source decay occur independent of the ionization process in matrix-assisted laser desorption?

The influence of the acidic and basic characters of constituent amino acid residues on the peptide fragment ions produced by in-source decay under matrix assisted laser desorption/ionization (MALDI) conditions has been studied using positive- and negative-ion experiments. Whereas the in-source decay spectra of peptides containing basic Arg and/or Lys residues near the N-terminus showed so-called c_n- and a_n-series ions in positive-ion mode, a peptide that has an acidic amino acid cluster near the N-terminus and a basic residue near the C-terminus characteristically formed y_n- and z_n-series ions in the positive-ion in-source decay spectrum. These results indicated that fragment ion series produced by in-source decay depend strongly upon the acidic and basic characters of the constituent amino acid residues and the near N- and C-termini. It was suggested that in-source decay processes occur intrinsically at NH–C^α and CO–NH bonds independent of the formation of molecular-related ions, and that the cleavages at the NH–C^α and CO–NH bonds occurred independently and were dependent on the matrix used.