计算机辅助的羧肽酶法测定多肽羧端顺序

在初步用计算机辅助的羧肽酶法测定了天花粉蛋白C-端顺序的基础上, 进一步设计了两个计算机程序-DPS程序和CPA程序. 用合成小肽和天然的肽对这两个计算机程序进行模型实验证明, 运用这两个程序能分别满意地从羧肽酶的酶解动力学曲线中获得重要的C-端顺序信息, 并测定了天花粉蛋白分子中未知肽段CB-3的C-端顺序为: -SerAlaSerAlaLeuHserOH, 这一顺序后来已经其他实验结果所证实. 本法不仅使C-端顺序测定延长至七个氨基酸, 而且还基本上解决了多肽或蛋白质含有多种多次重复氨基酸残基的C-端顺序测定.     

Predicting protein-protein interactions using graph invariants and a neural network

The PDZ domain of proteins mediates a protein-protein interaction by recognizing the hydrophobic C-terminal tail of the target protein. One of the challenges put forth by the DREAM (Discussions on Reverse Engineering Assessment and Methods) 2009 Challenge consists of predicting a position weight matrix (PWM) that describes the specificity profile of five PDZ domains to their target peptides. We consider the primary structures of each of the five PDZ domains as a numerical sequence derived from graph-theoretic models of each of the individual amino acids in the protein sequence. Using available PDZ domain databases to obtain known targets, the graph-theoretic based numerical sequences are then used to train a neural network to recognize their targets. Given the challenge sequences, the target probabilities are computed and a corresponding position weight matrix is derived. In this work we present our method. The results of our method placed second in the DREAM 2009 challenge.     

Simulation studies of the protein-water interface. I. Properties at the molecular resolution

We report molecular dynamics simulations of three globular proteins: ubiquitin, apo-calbindin D(9K), and the C-terminal SH2 domain of phospholipase C-gamma1 in explicit water. The proteins differ in their overall charge and fold type and were chosen to represent to some degree the structural variability found in medium-sized proteins. The length of each simulation was at least 15 ns, and larger than usual solvent boxes were used. We computed radial distribution functions, as well as orientational correlation functions about the surface residues. Two solvent shells could be clearly discerned about charged and polar amino acids. Near apolar amino acids the water density near such residues was almost devoid of structure. The mean residence time of water molecules was determined for water shells about the full protein, as well as for water layers about individual amino acids. In the dynamic properties, two solvent shells could be characterized as well. However, by comparison to simulations of pure water it could be shown that the influence of the protein reaches beyond 6 A, i.e., beyond the first two shells. In the first shell (r < or =3.5 A), the structural and dynamical properties of solvent waters varied considerably and depended primarily on the physicochemical properties of the closest amino acid side chain, with which the waters interact. By contrast, the solvent properties seem not to depend on the specifics of the protein studied (such as the net charge) or on the secondary structure element in which an amino acid is located. While differing considerably from the neat liquid, the properties of waters in the second solvation shell (3.5< r < or =6 A) are rather uniform; a direct influence from surface amino acids are already mostly shielded.     

Narrow-bore high-performance liquid chromatography of phenylthiocarbamyl amino acids and carboxypeptidase P digestion for protein C-terminal sequence analysis

Carboxypeptidase P digestion followed by narrow-bore high-performance liquid chromatography of phenylthiocarbamyl amino acids is employed for polypeptide C-terminal end group and sequence determination. Carboxypeptidase P digestion of polypeptides provides specific cleavage of protein C-terminal amino acids. The digestion offers the advantage that it can be carried out in either 10 mM sodium acetate or water at pH 4.0 in the presence of an enzyme activator, Brij-35. The narrow-bore high-performance liquid chromatography of all 20 phenylthiocarbamyl-amino acids has provided quantitative analysis at low picomole levels. This efficient and sensitive procedure is particularly useful for examining in vivo excision of protein C-termini and for verifying the integrity of various protein products produced by recombinant DNA techniques.     

Gas-liquid chromatographic analysis of protein amino acids as N-isobutyloxycarbonylamino acid methyl esters.

A practical method for the quantitative determination of protein amino acids by gas-liquid chromatography (GLC) is described. All of the common protein amino acids except arginine can be readily converted into their N-isobutyloxycarbonyl (N-isoBOC) methyl ester derivatives by a simple procedure involving isobutyloxycarbonylation with isobutyl chloroformate in aqueous medium, followed by methylation with diazomethane. Arginine was converted into N-isoBOC ornithine methyl ester by treatment with arginase, followed by the above derivatization procedure. The resulting N-isoBOC methyl esters of the amino acids have good GLC properties. Complete resolution of the derivatives of 20 protein amino acids was achieved by using a dual-column system consisting of a 0.65% Poly-A-101A column and a 0.70% FFAP-Poly-A-101A (1:1, w/w) column. The reproducibility of response was found to be good for derivatives carried through the entire chemical and chromatographic procedure. The calibration graphs were linear and showed no statistical bias. The results of recovery experiments with synthetic mixtures containing known amounts of the amino acids were satisfactory, the recoveries ranging from 94.3 to 106.2%.     

3D-Epitope-Explorer (3DEX): localization of conformational epitopes within three-dimensional structures of proteins

Neutralizing antibodies often recognize conformational, discontinuous epitopes. Linear peptides mimicking such conformational epitopes can be selected from phage display peptide libraries by screening with the respective antibodies. However, it is difficult to localize these "mimotopes" within the three-dimensional (3D) structures of the target proteins. Knowledge of conformational epitopes of neutralizing antibodies would help to design antigens able to elicit protective immune responses. Therefore, we provide here a software that allows to localize linear peptide sequences within 3D structures of proteins. The 3D-Epitope-Explorer (3DEX) software allows to map conformational epitopes in 3D protein structures based on an algorithm that takes into account the physicochemical neighborhood of C(alpha)- or C(beta)-atoms of individual amino acids. A given amino acid of a peptide sequence is localized within the protein and the software searches within predefined distances for the amino acids neighboring that amino acid in the peptide. Surface exposure of the amino acids can also be taken into consideration. The procedure is then repeated for the remaining amino acids of the peptide. The introduction of a joker function allows to map peptide mimotopes, which do not necessarily have 100% sequence homology to the protein. Using this software we were able to localize mimotopes selected from phage displayed peptide libraries with polyclonal antibodies from HIV-positive patient plasma within the 3D structure of gp120, the exterior glycoprotein of HIV-1. We also analyzed two recently published peptide sequences corresponding to known conformational epitopes to further confirm the integrity of 3DEX.     

A strategy for rapid and efficient sequencing of Lys-C peptides by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry post-source decay

A modification procedure for Lys-C peptides is described which simplifies the correct assignment of the amino acid sequence. Release of the C-terminal lysine from Lys-C peptides by carboxypeptidase B and subsequent N-terminal acetylation of the resulting peptides leads to predictable shifts of the C- and N-terminal fragment ions in Matrix-assisted laser desorption/ionisation time-of-flight post-source decay mass spectra and facilitates the correct assignment of mostly complete amino acid sequences for oligopeptides. The derived sequences of peptides from unknown proteins were used to search in databases for homologous protein sequences. Our method was applied to an unknown protein isolated from eggs of Drosophila melanogaster, resulting in the identification of a peptidyl prolyl cis-trans-isomerase.     

Spectral representation of reduced protein models

We consider a spectrum-like two-dimensional graphical representation of proteins based on a reduced protein model in which 20 amino acids are grouped into five classes. This particular grouping of amino acids was suggested by Riddle and co-workers in 1997. The graphical representation is based on depicting sequentially the amino acids on five horizontal lines at equal separations. One-letter codes, B, O, U, X and Y, to which numerical values 1 to 5 have been assigned, are suggested as labels for the fictional amino acids that represent all the amino acids within each group. The approach is illustrated on ND6 proteins of eight species having from 168 to 175 amino acids. While visual inspection of the novel spectral graphical representations of proteins may reveal local similarities and dissimilarities of protein sequences, arithmetic manipulations of spectra offer an elegant route to graphic visualization of the degree of similarity for selected pairs of proteins.     

An alignment-free method to find similarity among protein sequences via the general form of Chou’s pseudo amino acid composition

In this paper, we propose a method to create the 60-dimensional feature vector for protein sequences via the general form of pseudo amino acid composition. The construction of the feature vector is based on the contents of amino acids, total distance of each amino acid from the first amino acid in the protein sequence and the distribution of 20 amino acids. The obtained cosine distance metric (also called the similarity matrix) is used to construct the phylogenetic tree by the neighbour joining method. In order to show the applicability of our approach, we tested it on three proteins: 1) ND5 protein sequences from nine species, 2) ND6 protein sequences from eight species, and 3) 50 coronavirus spike proteins. The results are in agreement with known history and the output from the multiple sequence alignment program ClustalW, which is widely used. We have also compared our phylogenetic results with six other recently proposed alignment-free methods. These comparisons show that our proposed method gives a more consistent biological relationship than the others. In addition, the time complexity is linear and space required is less as compared with other alignment-free methods that use graphical representation. It should be noted that the multiple sequence alignment method has exponential time complexity.     

Structure of the mitochondrial URFA6L gene and tRNA(Lys) gene from CARP.

The carp mitochondrial URFA6L gene consists of 165 base pairs. The overall structural organization of the gene is very similar to that of the Xenopus URFA6L gene. Their nucleotide sequences exhibit 68% homology. The carp URFA6L gene encodes a protein of 54 amino acids. The amino acid composition of the protein is unusual because almost half of the residues consist of 5 hydrophobic amino acids (proline, tryptophan, leucine, isoleucine and tyrosine). A comparison between the amino acid sequences of 5 vertebrate URFA6L proteins and the yeast ATPase 8 showed that they have weak but very important common structural features, suggesting that the vertebrate URFA6L proteins may function as ATPase8. The nucleotide sequence of the lysine tRNA gene from carp has been determined and represented in clover-leaf secondary structure. Similar to amphibian and mammalian mitochondrial tRNA(Lys) genes, the carp mitochondrial tRNA(Lys) gene also has some unusual structural features as compared with its cytoplasmic counterpart. A comparison between the nucleotide sequences of the tRNA(Lys) gene from 7 vertebrates showed that the most conservative portions are the anticodon loop, nucleotides 8 and 9, the variable loop, the anticodon stem and the aminoacyl stem. The least conservative portions are the D-loop and the T-loop. These structural features may show that the mitochondrial tRNA(Lys) has a different interaction with mitochondrial ribosome.     

Automated phenylthiocarbamyl amino acid analysis of carboxypeptidase/aminopeptidase digests and acid hydrolysates

A fully automated exopeptidase digestion procedure for the partial determination of N- and C-terminal peptide/protein sequence is described. The digestion of various substrates with aminopeptidase M, carboxypeptidase A, P or Y was accomplished with the Varian 9090 autosampler's robotic automix routines. The released free amino acids, in addition to free amino acids from acid hydrolysates, were derivatized with phenylisothiocyanate in an automated fashion and subsequently chromatographed on a C18 column for separation and quantitation. The advantages of automating this precolumn phenylisothiocyanate derivatization are the virtual elimination of sample manipulation errors and very reproducible data due to the precise control of the reaction conditions both of which, facilitate the interpretation of the exopeptidase reaction kinetic data.     

The graphical representation of protein sequences based on the physicochemical properties and its applications

Based on the chaos game representation, a 2D graphical representation of protein sequences was introduced in which the 20 amino acids are rearranged in a cyclic order according to their physicochemical properties. The Euclidean distances between the corresponding amino acids from the 2‐D graphical representations are computed to find matching (or conserved) fragments of amino acids between the two proteins. Again, the cumulative distance of the 2D‐graphical representations is defined to compare the similarity of protein. And, the examination of the similarity among sequences of the ND5 proteins of nine species shows the utility of our approach. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2010     

Protein analysis by mass spectrometry and sequence database searching: tools for cancer research in the post-genomic era

The post-genomic era is characterized by the deposition of sequence information for entire genomes in databases. Currently, besides the protein sequences for known human proteins, there are partial sequences from thousands more human proteins for which no biological function has been assigned. A powerful new tool for the unambiguous identification and characterization of gel-separated proteins is accomplished by the combination of mass spectrometry and sequence database searching. This combination provides the cancer biologist with the ability to (i) identify the potential protein:protein associations and (ii) fully characterize function-critical post-translational modifications, both directly from silver-stained polyacrylamide gels. In this report we describe the application of tandem mass spectrometry and database searching to two problems which are prototypical for cancer research and indeed for biomedical research in general. The first is the identification of gel-separated, low abundance proteins based on amino acid sequence composition following coimmunoprecipitation with the human apoptosis inhibitor protein BclX(L). The second is the determination of the precise sites of phosphorylation of the human regulatory protein 4E-BP1, which controls mRNA translation.     

Massenspektren der von Aminosäuren bzw. Peptiden abgeleiteten Triazin-Derivate

A mass spectrometric study of about 30 triazine derivatives from amino acids and peptides is reported. These derivatives incorporated the C-terminal of amino acids and peptides in the ring. In contrast to the mass spectra of amino acids and peptide esters reported previously, they always showed characteristic fragments, denoting the presence of the terminal triazine ring. By using this peak as a marker, it is easy to estimate the C-terminal of peptides. In dipeptides, the N-terminal and C-terminal are determined simultaneously.     

Immobilized Carboxypeptidase A for Sequential Analysis

The use of carboxypeptidase for sequence determination is attractive because of its technical simplicity. In chemical methods all molecules of a peptide are made to go through a degradation cycle before a new cycle is started. In the enzymatic degradation of a protein, the order of the amino acid residues is not necessarily determined in a stepwise fashion but rather from the rate at which the amino acids appear in the digest, i.e., an amino acid appearing faster than another presumably precedes it in the sequence. Under favorable circumstances the rate of appearance of the amino acids released during digestion give sufficient evidence for the C-terminal sequence to be deduced. The present work pertains to a study on the use of immobilized carboxypeptidase A columns for sequential analyses.     

Spectrophotometric determination of cysteine and cystine in urine

A spectrophotometric method for the determination of cysteine and cystine in urine is described. This method is a modification of that reported for the determination of cysteine and cystine in proteins. The determination in urine is specific and simple. The colour develops at room temperature and no pre-treatment of urine is needed. Other amino acids, urea, uric acid, ascorbic acid, bilirubin, biliverdin, carbohydrates, hormones, acetone, salicylic acid, small amounts of protein and other common components of urine do not interfere. The method is particularly suitable for the diagnosis of hepatic cystinuria and other diseases characterised by high sulphur-containing amino acids in urine.     

The primary chemical structure of human milk lysozyme: establishment of a temporary formula

The detailed primary sequences of the N-terminal moiety (72 amino acid residues) and of the C-terminal end (23 amino acid residues) of human milk lysozyme (129 residues) are reported. A tentative complete structure of the enzyme is compared to hen and duck egg-white lysozymes which are very near related proteins despite many amino acid replacements (around 50), the insertion of an additional residue in the N-terminal and a deletion in the C-terminal moiety.     

Artificial chemokines: combining chemistry and molecular biology for the elucidation of interleukin-8 functionality

How can we understand the contribution of individual parts or segments to complex structures? A typical strategy to answer this question is simulation of a segmental replacement followed by realization and investigation of the resulting effect in structure-activity studies. For proteins, this problem is commonly addressed by site-directed mutagenesis. A more general approach represents the exchange of whole secondary structure elements by rationally designed segments. For a demonstration of this possibility we identified the alpha-helix at the C-terminus of human interleukin-8 (hIL-8). Since this chemokine possesses four conserved cysteine residues, it can easily be altered by ligation strategies. A set of different segments, which are able to form amphiphilic helices, was synthesized to mimic the C-terminal alpha-helix. Beside sequences of alpha-amino acids, oligomers of non-natural beta(3)-amino acids with the side chains of canonical amino acids were introduced. Such beta-peptides form helices, which differ from the alpha-helix in handedness and dipole orientation. Variants of the semisynthetic hIL-8 proteins demonstrated clearly that the exact side chain orientation is of more importance than helix handedness and dipole orientation. The activity of a chimeric protein with a beta-peptide helix that mimics the side chain orientation of the native alpha-helix most perfectly is comparable to that of the native hIL-8. Concepts like this could be a first step toward the synthesis of proteins consisting of large artificial secondary structure elements.     

Seed proteins of Ricinus communis. III. Isolation and characterization of the two-component polypeptide chains of acid resins

It has been shown that the acid form of ricin — ricin T — consists of two nonidentical subunits: isoleucine and alanine subunits with molecular weights of 30 and 28 kDa, respectively. The isoelectric points, N- and C-terminal amino acids, and partial amino acid sequences of these subunits have been determined. The two subunits of ricin T have been found to exhibit differences from and similarities to ricins D and E.