Prediction and analysis of protein hydroxyproline and hydroxylysine

Background

Hydroxylation is an important post-translational modification and closely related to various diseases. Besides the biotechnology experiments, in silico prediction methods are alternative ways to identify the potential hydroxylation sites.

Methodology/Principal Findings

In this study, we developed a novel sequence-based method for identifying the two main types of hydroxylation sites – hydroxyproline and hydroxylysine. First, feature selection was made on three kinds of features consisting of amino acid indices (AAindex) which includes various physicochemical properties and biochemical properties of amino acids, Position-Specific Scoring Matrices (PSSM) which represent evolution information of amino acids and structural disorder of amino acids in the sliding window with length of 13 amino acids, then the prediction model were built using incremental feature selection method. As a result, the prediction accuracies are 76.0% and 82.1%, evaluated by jackknife cross-validation on the hydroxyproline dataset and hydroxylysine dataset, respectively. Feature analysis suggested that physicochemical properties and biochemical properties and evolution information of amino acids contribute much to the identification of the protein hydroxylation sites, while structural disorder had little relation to protein hydroxylation. It was also found that the amino acid adjacent to the hydroxylation site tends to exert more influence than other sites on hydroxylation determination.

Conclusions/Significance

These findings may provide useful insights for exploiting the mechanisms of hydroxylation.     

Discarding functional residues from the substitution table improves predictions of active sites within three-dimensional structures

Substitutions of individual amino acids in proteins may be under very different evolutionary restraints depending on their structural and functional roles. The Environment Specific Substitution Table (ESST) describes the pattern of substitutions in terms of amino acid location within elements of secondary structure, solvent accessibility, and the existence of hydrogen bonds between side chains and neighbouring amino acid residues. Clearly amino acids that have very different local environments in their functional state compared to those in the protein analysed will give rise to inconsistencies in the calculation of amino acid substitution tables. Here, we describe how the calculation of ESSTs can be improved by discarding the functional residues from the calculation of substitution tables. Four categories of functions are examined in this study: protein–protein interactions, protein–nucleic acid interactions, protein–ligand interactions, and catalytic activity of enzymes. Their contributions to residue conservation are measured and investigated. We test our new ESSTs using the program CRESCENDO, designed to predict functional residues by exploiting knowledge of amino acid substitutions, and compare the benchmark results with proteins whose functions have been defined experimentally. The new methodology increases the Z-score by 98% at the active site residues and finds 16% more active sites compared with the old ESST. We also find that discarding amino acids responsible for protein–protein interactions helps in the prediction of those residues although they are not as conserved as the residues of active sites. Our methodology can make the substitution tables better reflect and describe the substitution patterns of amino acids that are under structural restraints only.     

Uptake of Lysine by Wild-Type and S-(2-Aminoethyl)-L-cysteine-Resistant Suspension-Cultured Cells of Triticum aestivum

The kinetics of uptake of L-lysine in wheat (Triticum aestivumcv. Chinese Spring) were analyzed in wild-type cells and inAEC-1 variant cells that are resistant to S-(2-aminoethyl)-L-cysteine(AEC). Uptake of lysine by AEC-1 cells was considerably slowerthan that by the wild-type cells. In the presence of carbonylcyanidem-chlorophenylhydrazone, the rates of uptake by both types ofcell were reduced to a similar linear component. Fitting theuptake data to one linear (diffusional) component and one Michaelis-Menten(active) system showed that, as compared to wild-type cells,AEC-1 cells have a reduced V_max and an increased K_m with respectto the active component, byt they have a similar diffusionalcomponent. Inhibition experiments with various amino acids indicatedthat the active component represents a carrier specific forbasic amino acids, which was competitively inhibited by AEC.The AEC-1 cells also showed reduced uptake of several neutraland acidic amino acids, but the rate of uptake of 3-O-methylglucosewas somewhat higher than that by wild-type cells. (Received May 16, 1989; Accepted September 4, 1989)     

The stereochemical resolution of enantiomeric free and derivatized amino acids using an HPLC chiral stationary phase based on immobilized alpha-chymotrypsin: chiral separation due to solute structure or enzyme activity

The stereochemical separation of free and derivatized amino acids on active alpha-chymotrypsin bonded to silica is governed by two mechanisms based on the structure of the solutes or on the enzymatic activity of the enzyme. The deactivation of the hydrolytically active site of the enzyme demonstrated that a significant portion of the retention on this support is due to hydrophobic interactions at other sites. These sites appear to be stereoselective for the ester derivatives of amino acids but not for the other studied solutes.     

A general DNA analysis program for the Hewlett-Packard Model 86/87 microcomputer.

A program is described to perform general DNA sequence analysis on the Hewlett-Packard Model 86/87 microcomputer operating on 128 K of RAM. The following analytical procedures can be performed: 1. display of the sequence, in whole or part, or its complement; 2. search for specified sequences e.g. restriction sites, and in the case of the latter give fragment sizes; 3. perform a comprehensive search for all known restriction enzyme sites; 4. map sites graphically; 5. perform editing functions; 6. base frequency analysis; 7. search for repeated sequences; 8. search for open reading frames or translate into the amino acid sequence and analyse for basic and acidic amino acids, hydrophobicity, and codon usage. Two sequences, or parts thereof, can be merged in various orientations to mimic recombination strategies, or can be compared for homologies. The program is written in HP BASIC and is designed principally as a tool for the laboratory investigator manipulating a defined set of vectors and recombinant DNA constructs.     

Variation in structural location and amino acid conservation of functional sites in protein domain families

Background  

The functional sites of a protein present important information for determining its cellular function and are fundamental in drug design. Accordingly, accurate methods for the prediction of functional sites are of immense value. Most available methods are based on a set of homologous sequences and structural or evolutionary information, and assume that functional sites are more conserved than the average. In the analysis presented here, we have investigated the conservation of location and type of amino acids at functional sites, and compared the behaviour of functional sites between different protein domains.     

Unnatural amino acid mutagenesis: a precise tool for probing protein structure and function

The first general method for the biosynthetic incorporation of unnatural amino acids into proteins was reported in 1989. The ensuing years have seen the solid development and subsequent implementation of "unnatural amino acid mutagenesis" in a number of groundbreaking studies. Over 100 different amino acids have been incorporated into dozens of soluble and transmembrane proteins, using both cell-extract and cell-intact translation systems. The approach has provided insights into ligand-binding sites, conformational changes, and protein-protein interactions with a level of precision simply unparalleled by conventional mutagenesis. Here, the methodology is outlined, significant applications of the approach are summarized, and recent major improvements in the method are discussed. The future will likely see many more investigators utilizing this approach to manipulate proteins as it realizes its promise of becoming a tool with enormous potential.     

Loop engineering of amadoriase II and mutational cooperativity

Amadori compounds and their cross-linked products have been implicated in diabetic complications and some age-related diseases. Fructosyl amine oxidases (FAOXs) are a family of enzymes that can cleave the amadori compounds. However, the natural enzymes are only active on small substrates (fructosyl amino acids or dipeptides), which limits the therapeutic and diagnostic applications of these enzymes. In this study, amadoriase II, a member of the FAOX family from Aspergillus fumigatus was engineered to broaden its substrate range using a modified combinatorial active site saturation testing approach. The two loops at the entrance of the substrate channel were targeted. Saturation mutagenesis was carried out to search for hot-spot sites, followed by pairwise mutagenesis and subsequent combination of active mutations. Five sites on the loops were found to be critical for accessibility for two model bulky substrates, fructosyl adamantanamine and fructosyl-polylysine (3–13 lysines). Two best mutants (with three and five mutations, respectively) were obtained, with a specific activity toward the model substrates 20.6-fold and 16.8-fold that of the wild-type, respectively. Deconvolution experiments revealed the cooperativity of the mutations.     

Critical amino acids in the active site of meprin metalloproteinases for substrate and peptide bond specificity

The protease domains of the evolutionarily related alpha and beta subunits of meprin metalloproteases are approximately 55% identical at the amino acid level; however, their substrate and peptide bond specificities differ markedly. The meprin beta subunit favors acidic residues proximal to the scissile bond, while the alpha subunit prefers small or aromatic amino acids flanking the scissile bond. Thus gastrin, a peptide that contains a string of five Glu residues, is an excellent substrate for meprin beta, while it is not hydrolyzed by meprin alpha. Work herein aimed to identify critical amino acids in the meprin active sites that determine the substrate specificity differences. Sequence alignments and homology models, based on the crystal structure of the crayfish astacin, showed electrostatic differences within the meprin active sites. Site-directed mutagenesis of active site residues demonstrated that replacement of a hydrophobic residue by a basic amino acid enabled the meprin alpha protease to cleave gastrin. The meprin alphaY199K mutant was most effective; the corresponding mutation of meprin betaK185Y resulted in decreased activity toward gastrin. Peptide cleavage site determinations and kinetic analyses using a variety of peptides extended evidence that meprin alphaTyr-199/betaLys-185 are substrate specificity determinants in meprin active sites. These studies shed light on the molecular basis for the substrate specificity differences of astacin metalloproteinases.     

L-海因酶与D-海因酶的序列及结构比较研究

运用生物信息学的研究方法,从序列及结构上对L型及D型海因酶进行了初步的比较。研究了两种类型的海因酶在序列、骨架结构及活性中心的区别,并探讨了产生这些差异的理论基础,为海因酶进一步的理论及应用研究提供一定的指导。     

Identification of Des-methyl-DIF-1 Methyltransferase in Dictyostelium purpureum

For the production of D-amino acids using stable N-carbamyl-D-amino acid amidohydrolase (DCase) in an immobilized form, the DCase gene of Agrobacterium sp. KNK712 was mutagenized to increase its enzymatic thermostability. In a search for thermostability-related amino acid sites besides the two known sites of DCase, i.e., the 57th and 203rd amino acids, the new mutant enzyme found, in which the 236th amino acid, valine, had been changed to alanine, showed a 10°C increase in thermostability. These known three thermostability-related amino acids were changed to other amino acids by the PCR technique, and it was proved that the thermostability of the DCase increased when the 57th amino acid of DCase, histidine, was changed to leucine, the 203rd amino acid, proline, to asparagine, glutamate, alanine, isoleucine, histidine, or threonine, and the 236th amino acid, valine, to threonine or serine, in addition to the known mutations.     

Identification of phosphorylation sites for adenosine 3',5'-cyclic phosphate dependent protein kinase on the voltage-sensitive sodium channel from Electrophorus electricus

The voltage-sensitive sodium channel from the electroplax of Electrophorus electricus is selectively phosphorylated by the catalytic subunit of cyclic-AMP-dependent protein kinase (protein kinase A) but not by protein kinase C. Under identical limiting conditions, the protein was phosphorylated 20% as rapidly as the synthetic model substrate kemptamide. A maximum of 1.7 +/- 0.6 equiv of phosphate is incorporated per mole. Phosphoamino acid analysis revealed labeled phosphoserine and phosphothreonine at a constant ratio of 3.3:1. Seven distinct phosphopeptides were identified among tryptic fragments prepared from radiolabeled, affinity-purified protein and resolved by HPLC. The three most rapidly labeled fragments were further purified and sequenced. Four phosphorylated amino acids were identified deriving from three consensus phosphorylation sites. These were serine 6, serine 7, and threonine 17 from the amino terminus and a residue within 47 amino acids of the carboxyl terminus, apparently serine 1776. The alpha-subunits of brain sodium channels, like the electroplax protein, are readily phosphorylated by protein kinase A. However, these are also phosphorylated by protein kinase C and exhibit a markedly different pattern of incorporation. Each of three brain alpha-subunits displays an approximately 200 amino acid segment between homologous repeat domains I and II, which is missing from the electroplax and skeletal muscle proteins [Noda et al. (1986) Nature (London) 320, 188; Kayano et al. (1988) FEBS Lett. 228, 1878; Trimmer et al. (1989) Neuron 3, 33]. Most of the phosphorylation of the brain proteins occurs on a cluster of consensus phosphorylation sites located in this segment. This contrasts with the pattern of highly active sites on the amino and carboxyl termini of the electroplax protein. The detection of seven labeled tryptic phosphopeptides compared to the maximal labeling stoichiometry of approximately 2 suggests that many of the acceptor sites on the protein may be blocked by endogenous phosphorylation.     

Microwave-assisted nonspecific proteolytic digestion and controlled methylation for glycomics applications

Nonspecific proteolytic digestion of glycoproteins is an established technique in glycomics and glycoproteomics. In the presence of pronase E, for example, glycoproteins are digested to small glycopeptides having one to six amino acids residues, which can be analyzed with excellent sensitivity using mass spectrometry. Unfortunately, the long digestion times (1-3 days) limit the analytical throughput. In this study, we used controlled microwave irradiation to accelerate the proteolytic cleavage of glycoproteins mediated by pronase E. We used ESI-MS and MALDI-MS analyses to evaluate the microwave-assisted enzymatic digestions at various digestion durations, temperatures, and enzyme-to-protein ratios. When digesting glycoproteins, pronase E produced glycopeptides within 5 min under microwave irradiation; glycopeptides having one or two amino acids were the major products. Although analysis of peptides containing multiple amino acid residues offers the opportunity for peptide sequencing and provides information regarding the sites of glycosylation, the signals of Asn-linked glycans were often suppressed by the glycopeptides containing basic amino acids (Lys or Arg) in MALDI-MS experiments. To minimize this signal-to-content dependence, we converted the glycopeptides into their sodiated forms and then methylated them using methyl iodide. This controlled methylation procedure resulted in quaternization of the amino group of the N-terminal amino acid residue. Using this approach, the mass spectrometric response of glyco-Asn was enhanced, compensating for the poorer ionization efficiency associated with the basic amino acids residues. The methylated products of glycopeptides containing two or more amino acid residues were more stable than those containing only a single Asn residue. This feature can be used to elucidate glycan structures and glycosylation sites without the need for MS/MS analysis.     

Chemoreception of amino acids in larvae of two species of Pieris

ABSTRACT. Specificity and sensitivity of gustatory neurones in response to twenty-two amino acids were studied in larvae of Pieris brassicae L. and Pieris rapae L. (Lepidoptera: Pieridae) using electrophysiological methods. Twelve amino acids stimulated a specific amino acid receptor cell in the lateral styloconic sensillum on the maxillary galea of both species, and a further two evoked single unit responses in the same sensillum of P.brassicae only. Histidine, phenylalanine and tryptophane were the weakest stimulants for P.brassicae , but were among the four best stimulants for P.rapae . In both species, eight amino acids were ineffective. Significant differences in stimulatory effectiveness were found between amino acids. Nutritionally essential amino acids were more effective in both species, as in five other lepidopterous species. Similarities with postulated sites for amino acid recognition in the dipteran Boettcherisca peregrina were found.
Concentration-response (C/R) relations were studied for five amino acids. Significant differences were found in saturated response levels. Parameters characterizing C/R relations were estimated using a logistic model. Comparing C/R parameters with phytochemical data on concentrations of free amino acids in a common host plant, Brassica oleracea L., shows that amino acids are effective stimuli at their natural concentrations. The amino acid chemoreceptor seems able to transmit information about concentration differences of amino acids in the plant tissue.     

Guiding conformation space search with an all-atom energy potential

The most significant impediment for protein structure prediction is the inadequacy of conformation space search. Conformation space is too large and the energy landscape too rugged for existing search methods to consistently find near-optimal minima. To alleviate this problem, we present model-based search, a novel conformation space search method. Model-based search uses highly accurate information obtained during search to build an approximate, partial model of the energy landscape. Model-based search aggregates information in the model as it progresses, and in turn uses this information to guide exploration toward regions most likely to contain a near-optimal minimum. We validate our method by predicting the structure of 32 proteins, ranging in length from 49 to 213 amino acids. Our results demonstrate that model-based search is more effective at finding low-energy conformations in high-dimensional conformation spaces than existing search methods. The reduction in energy translates into structure predictions of increased accuracy.     

Amino acids as taste stimuli for tsetse flies

Abstract.This paper reports the responses of taste cells on the legs of the blood-feeding tsetse fly Glossina fuscipes fuscipes Newstead 1910 (Diptera: Glossinidae) to twenty protein amino acids and to their mixture as it is present in human sweat. It is investigated whether the mixture is sensed differently than the amino acids singly. The taste cells are most sensitive to phenylalanine (K≈ 1 μm ) and tyrosine; and they respond in a lesser degree to methionine, valine, isoleucine, cysteine, tryptophan, histidine, alanine, and threonine. The amino acids serine, proline, asparagine, arginine, glutamine, lysine, aspartic acid, glutamic acid, and glycine give little or no response even at 10 mm . As the succession of effectiveness of the amino acids appears to be the same for all cells, it is deduced that the flies are unable to discriminate the amino acids by comparing responses across sensory cells. A temporal coding of quality does not seem feasible either. Thus, the properties of the taste cells limit the sense to assessing the intensity of an amino acid stimulus and not its identity. Although several parameters in the response adaptation curves are concentration-dependent, it is suggested that the flies judge intensity of a stimulus only from the first 50 or so milliseconds. Although other studies and these indicate that a multiplicity of binding sites may be responsible for the reception of amino acids, the response to the mixture can be predicted from a no-interaction model, whereby each ligand's access to the binding sites is proportional to its mole fraction. It is argued that this may be the case for more of the naturally occurring mixtures which comprise structurally similar ligands. The responses to the mixture and to phenylalanine alone are equally susceptible to inhibition by sodium chloride. It is suggested that, although discrimination of hosts probably requires another sense, the sense of taste is an excellent tool to detect a host underfoot during the local search for a feeding site.