N-terminal N-myristoylation of proteins: prediction of substrate proteins from amino acid sequence

Myristoylation by the myristoyl-CoA:protein N-myristoyltransferase (NMT) is an important lipid anchor modification of eukaryotic and viral proteins. Automated prediction of N-terminal N-myristoylation from the substrate protein sequence alone is necessary for large-scale sequence annotation projects but it requires a low rate of false positive hits in addition to a sufficient sensitivity.Our previous analysis of substrate protein sequence variability, NMT sequences and 3D structures has revealed motif properties in addition to the known PROSITE motif that are utilized in a new predictor described here. The composite prediction function (with separate ad hoc parameterization (a) for queries from non-fungal eukaryotes and their viruses and (b) for sequences from fungal species) consists of terms evaluating amino acid type preferences at sequences positions close to the N terminus as well as terms penalizing deviations from the physical property pattern of amino acid side-chains encoded in multi-residue correlation within the motif sequence. The algorithm has been validated with a self-consistency and two jack-knife tests for the learning set as well as with kinetic data for model substrates. The sensitivity in recognizing documented NMT substrates is above 95 % for both taxon-specific versions. The corresponding rate of false positive prediction (for sequences with an N-terminal glycine residue) is close to 0.5 %; thus, the technique is applicable for large-scale automated sequence database annotation. The predictor is available as public WWW-server with the URL http://mendel.imp.univie.ac.at/myristate/. Additionally, we propose a version of the predictor that identifies a number of proteolytic protein processing sites at internal glycine residues and that evaluates possible N-terminal myristoylation of the protein fragments.A scan of public protein databases revealed new potential NMT targets for which the myristoyl modification may be of critical importance for biological function. Among others, the list includes kinases, phosphatases, proteasomal regulatory subunit 4, kinase interacting proteins KIP1/KIP2, protozoan flagellar proteins, homologues of mitochondrial translocase TOM40, of the neuronal calcium sensor NCS-1 and of the cytochrome c-type heme lyase CCHL. Analyses of complete eukaryote genomes indicate that about 0.5 % of all encoded proteins are apparent NMT substrates except for a higher fraction in Arabidopsis thaliana ( approximately 0.8 %).     

Extracting information on folding from the amino acid sequence: consensus regions with preferred conformation in homologous proteins.

It is investigated whether protein segments predicted to have a well-defined conformational preference in the absence of tertiary interactions are conserved in families of homologous proteins. The prediction method follows the procedures of Rooman, M., Kocher, J.-P., and Wodak, S. (preceding paper in this issue). It uses a knowledge-based force field that incorporates only local interactions along the sequence and identifies segments whose lowest energy structure displays a sizable energy gap relative to other computed conformations. In 13 of the protein families and subfamilies considered that are sufficiently homologous to have similar 3D structures, at least one region is consistently predicted as having the same preferred conformation in virtually all family members. These regions are between 4 and 26 residues long. They are often located at chain ends and correspond primarily to segments of secondary structure heavily involved in interactions with the rest of the protein, suggesting that they could act as nuclei around which other parts of the structure would assemble. Experimental data on early folding intermediates or on protein fragments with appreciable structure in aqueous solution are available for more than half of the protein families. Comparison of our results with these data is quite favorable. They reveal that each of the experimentally identified early formed, or independently stable, substructures harbors at least one of the segments consistently predicted as having a preferred conformation by our procedure. The implications of our findings for the conservation of folding pathways in homologous proteins are discussed.     

Membrane proteins: amino acid sequence and membrane penetration

A computer study shows that the membrane-penetrating portion of the erythrocyte surface MN-glycoprotein (Winzler, 1969; Marchesi et al., 1972) is distinguishable by informal cluster analysis from other segments of globular proteins when sequence length is plotted against hydrophobicity This analysis further suggests the possibility that other membrane-penetrating segments of proteins can be identified in the same way.     

The complete amino acid sequence of the human erythrocyte membrane anion-transport protein deduced from the cDNA sequence.

1. We have isolated cDNA clones corresponding to the red cell membrane anion-transport protein (Band 3). 2. The cDNA clones cover 3475 bases of the mRNA and contain the entire protein-coding region, 150 bases of the 5' untranslated region and part of the 3' non-coding region, but do not extend to the 3' end of the mRNA. 3. The translated protein sequence predicts that the human red cell anion transporter contains 911 amino acids. 4. The availability of the amino acid sequence allows the interpretation of some of the many studies on the chemical and proteolytic modification of the human protein aimed at examining the structure and mechanism of this membrane transport protein.     

利用伪氨基酸组分预测分枝杆菌膜蛋白类型

膜蛋白是重要的药物靶位点,对膜蛋白类型的研究有助于药物的成功设计,因此正确预测膜蛋白类型对于药物研发是十分必要的。本文采用由274条分枝杆菌膜蛋白序列组成的一致性小于40%的数据集,以经过优化的伪氨基酸组分为特征,利用支持向量机分类算法预测分枝杆菌膜蛋白类型,在Jackknife检验下,得到85.4%的总体准确率和72.2%的平均准确率。结果说明,该方法可用于分枝杆菌膜蛋白类型的识别,将有助于抗分枝杆菌药物的开发。     

Topology prediction of membrane proteins.

A new method is described for prediction of protein membrane topology (intra- and extracellular sidedness) from multiply aligned amino acid sequences after determination of the membrane-spanning segments. The prediction technique relies on residue compositional differences in the protein segments exposed at each side of the membrane. Intra/extracellular ratios are calculated for the residue types Asn, Asp, Gly, Phe, Pro, Trp, Tyr, and Val, preferably found on the extracellular side, and for Ala, Arg, Cys, and Lys, mostly occurring on the intracellular side. The consensus over these 12 residue distributions is used for sidedness prediction. The method was developed with a test set of 42 protein families, for which all but one were correctly predicted with the new algorithm. This represents an improvement over predictions based on the widely used "positive-inside rule" and other techniques, where at least six mispredictions were observed for the same data set. Further, application of this and other methods to 12 protein families not in the test set still showed the better performance of the present technique, which was subsequently applied to another set of membrane protein families where the topology has yet to be determined.     

A possible way for prediction of domain boundaries in globular proteins from amino acid sequence

A simple approach to domain border prediction in globular proteins is outlined relying on the amino acid sequence only. Statistically determined sequential and association preference data of amino acids were combined to generate short range preference profiles along the polypeptide chains. Domain boundaries correlate with the minima of preference profiles, but some false minima also exist. Possibilities are discussed to exclude the false minima and to further improve the efficiency of the algorithm.     

The exchangeability of amino acids in proteins

The comparative analysis of protein sequences depends crucially on measures of amino acid similarity or distance. Many such measures exist, yet it is not known how well these measures reflect the operational exchangeability of amino acids in proteins, since most are derived by methods that confound a variety of effects, including effects of mutation. In pursuit of a pure measure of exchangeability, we present (1) a compilation of data on the effects of 9671 amino acid exchanges engineered and assayed in a set of 12 proteins; (2) a statistical procedure to combine results from diverse assays of exchange effects; (3) a matrix of "experimental exchangeability" values EX(ij) derived from applying this procedure to the compiled data; and (4) a set of three tests designed to evaluate the power of an exchangeability measure to (i) predict the effects of amino acid exchanges in the laboratory, (ii) account for the disease-causing potential of missense mutations in the human population, and (iii) model the probability of fixation of missense mutations in evolution. EX not only captures useful information on exchangeability while remaining free of other effects, but also outperforms all measures tested except for the best-performing alignment scoring matrix, which is comparable in performance.     

Predicting surface exposure of amino acids from protein sequence

The amino acid residues on a protein surface play a key role in interaction with other molecules, determined many physical properties, and constrain the structure of the folded protein. A database of monomeric protein crystal structures was used to teach computer-simulated neural networks rules for predicting surface exposure from local sequence. These trained networks are able to correctly predict surface exposure for 72% of residues in a testing set using a binary model, (buried/exposed) and for 54% of residues using a ternary model (buried/intermediate/exposed). In the ternary model, only 11% of the exposed residues are predicted as buried and only 5% of the buried residues are predicted as exposed. Also, since the networks are able to predict exposure with a quantitative confidence estimate, it is possible to assign exposure for over half of the residues in a binary model with greater than 80% accuracy. Even more accurate predictions are obtained by making a consensus prediction of exposure for a homologous family. The effect of the local environment of an amino acid on its accessibility, though smaller than expected, is significant and accounts for the higher success rate of prediction than obtained with previously used criteria. In the absence of a three-dimensional structure, the ability to predict surface accessibility of amino acids directly from the sequence is a valuable tool in choosing sites of chemical modification or specific mutations and in studies of molecular interaction.     

COPS--cis/trans peptide bond conformation prediction of amino acids on the basis of secondary structure information

SUMMARY: COPS predicts for all 20 naturally occurring amino acids whether the peptide bond in a protein is in cis or trans conformation. The algorithm is based only on secondary structure information of amino acid triplets without considering the amino acid sequence information. Conformation parameters are derived from solved 3D structures deposited in the PDB and led to propensities based on modified Chou-Fasman parameters. COPS analyses amino acid triplets taking only their respective secondary structure into consideration and upon application of a set of rules utilizing the conformation parameters, the N-terminal peptide bond conformation of the middle residue is predicted. COPS was tested on a random selection of protein datasets. AVAILABILITY: The COPS program and further information are freely available from the FMP website at http://www.fmp-berlin.de/nmr/cops CONTACT: labudde@fmp-berlin.de.     

An improved chemical approach toward the C-terminal sequence analysis of proteins containing all natural amino acids.

An improved chemical method, capable of derivatizing all natural amino acids to their corresponding thiohydantoins, is described. This involves activation by acetyl chloride in TFA followed by derivatization with ammonium thiocyanate. Possible interference of reactive side chains was investigated by reacting N-acetylamino acids as well as several peptides with propionyl chloride instead of acetyl chloride. The products were characterized by PDMS mass spectrometry and 1H-NMR. This chemical method allows, for the first time, complete derivatization of N-acetylproline to proline thiohydantoin. Applying this chemistry to peptides with a C-terminal proline, the yields for formation of proline thiohydantoin were found to be up to 60%, depending on the peptide sequence. The previous inability to derivatize C-terminal proline to thiohydantoin was thought to stem from the fact that proline cannot form the oxazolonium ion required for efficient reaction with the thiocyanate ion. However, we have found mass spectrometric evidence for the existence of a proline oxazolonium ion, under basic as well as under acidic conditions. This improvement in derivatization of C-terminal amino acids including proline is a major step forward in the development of a general chemical C-terminal sequencing method that permits the C-terminal sequence analysis of proteins of any amino acid composition.