Acid Residues In Proteins Research Articles

Critical amino acid residues in proteins: a BioMart integration of Reactome protein annotations with PRIDE mass spectrometry data and COSMIC somatic mutations

The reversible phosphorylation of serine, threonine and tyrosine hydroxyl groups is an especially prominent form of post-translational modification (PTM) of proteins. It plays critical roles in the regulation of diverse processes, and mutations that directly or indirectly affect these phosphorylation events have been associated with many cancers and other pathologies. Here, we describe the development of a new BioMart tool that gathers data from three different biological resources to provide the user with an integrated view of phosphorylation events associated with a human protein of interest, the complexes of which the protein (modified or not) is a part, the reactions in which the protein and its complexes participate and the somatic mutations that might be expected to perturb those functions. The three resources used are the Reactome, PRIDE and COSMIC databases. The Reactome knowledgebase contains annotations of phosphorylated human proteins linked to the reactions in which they are phosphorylated and dephosphorylated, to the complexes of which they are parts and to the reactions in which the phosphorylated proteins participate as substrates, catalysts and regulators. The PRIDE database holds extensive mass spectrometry data from which protein phosphorylation patterns can be inferred, and the COSMIC database holds records of somatic mutations found in human cancer cells. This tool supports both flexible, user-specified queries and standard (‘canned’) queries to retrieve frequently used combinations of data for user-specified proteins and reactions. We demonstrate using the Wnt signaling pathway and the human c-SRC protein how the tool can be used to place somatic mutation data into a functional perspective by changing critical residues involved in pathway modulation, and where available, check for mass spectrometry evidence in PRIDE supporting identification of the critical residue.Database URL: http://www.reactome.org/cgi-bin/mart

Recent advances in the photochemical control of protein function

Biological processes are regulated with a high level of spatial and temporal resolution. To understand and manipulate these processes, scientists need to be able to regulate them with Nature's level of precision. In this context, light is a unique regulatory element because it can be precisely controlled in terms of location, timing and amplitude. Moreover, most biological laboratories have a wide range of light sources as standard equipment. This review article summarizes the most recent advances in light-mediated regulation of protein function and its application in a cellular context. Specifically, the photocaging of small-molecule modulators of protein function and of specific amino acid residues in proteins is discussed. In addition, examples of the photochemical control of protein function through the application of genetically engineered natural-light receptors are presented.

Elucidating mechanisms of chlorine toxicity: reaction kinetics, thermodynamics, and physiological implications.

Industrial and transport accidents, accidental releases during recreational swimming pool water treatment, household accidents due to mixing bleach with acidic cleaners, and, in recent years, usage of chlorine during war and in acts of terror, all contribute to the general and elevated state of alert with regard to chlorine gas. We here describe chemical and physical properties of Cl(2) that are relevant to its chemical reactivity with biological molecules, including water-soluble small-molecular-weight antioxidants, amino acid residues in proteins, and amino-phospholipids such as phosphatidylethanolamine and phosphatidylserine that are present in the lining fluid layers covering the airways and alveolar spaces. We further conduct a Cl(2) penetration analysis to assess how far Cl(2) can penetrate the surface of the lung before it reacts with water or biological substrate molecules. Our results strongly suggest that Cl(2) will predominantly react directly with biological molecules in the lung epithelial lining fluid, such as low-molecular-weight antioxidants, and that the hydrolysis of Cl(2) to HOCl (and HCl) can be important only when these biological molecules have been depleted by direct chemical reaction with Cl(2). The results from this theoretical analysis are then used for the assessment of the potential benefits of adjuvant antioxidant therapy in the mitigation of lung injury due to inhalation of Cl(2) and are compared with recent experimental results.

Multivariate analysis of properties of amino acid residues in proteins from a viewpoint of functional site prediction

For the prediction of a protein’s function from its 3D-structure alone, it is of importance to elucidate by which properties functional site residues in a protein are discriminated from other residues. Here, we calculated five kinds of geometrical or physical properties of each residue in a protein. Those properties were integrated with techniques of multivariate analysis such as principal component analysis (PCA) or kernel PCA. Consequently, functional residues were found to show some distinct distributions in the scatter plot of those integrated data, which led to the proposal of a method for functional site prediction with a good performance.

PH-Dependent pKa Values in Proteins—A Theoretical Analysis of Protonation Energies with Practical Consequences for Enzymatic Reactions

Because of their central importance for understanding enzymatic mechanisms, pK(a) values are of great interest in biochemical research. It is common practice to determine pK(a) values of amino acid residues in proteins from NMR or FTIR titration curves by determining the pH at which the protonation probability is 50%. The pH dependence of the free energy required to protonate this residue is then determined from the linear relationship DeltaG(prot) = RT ln 10 (pH-pK(a)), where R is the gas constant and T the absolute temperature. However, this approach neglects that there can be important electrostatic interactions in the proteins that can shift the protonation energy. Even if the titration curves seem to have a standard sigmoidal shape, the protonation energy of an individual site in a protein may depend nonlinearly on pH. To account for this nonlinear dependence, we show that it is required to introduce pK(a) values for individual sites in proteins that depend on pH. Two different definitions are discussed. One definition is based on a rearranged Henderson-Hasselbalch equation, and the other definition is based on an equation that was used by Tanford and Roxby to approximate titration curves of proteins. In the limiting case of weak interactions, the two definitions lead to nearly the same pK(a) value. We discuss how these two differently defined pK(a) values are related to the free energy change required to protonate a site. Using individual site pK(a) values, we demonstrate on simple model systems that the interactions between protonatable residues in proteins can help to maintain the energy required to protonate a site in the protein nearly constant over a wide pH range. We show with the example of RNase T1 that such a mechanism to keep the protonation energy constant is used in enzymes. The pH dependence of pK(a) values may be an important concept in enzyme catalysis. Neglecting this concept, important features of enzymes may be missed, and the enzymatic mechanism may not be fully understood.

Role of aromatic amino acid tryptophan UVA-photoproducts in the determination of drug photosensitization mechanism: a comparison between methylene blue and naproxen

The aromatic amino acid tryptophan is the most susceptible protein residue involved in various photosensitized adverse effects. Of these processes, the tryptophan photosensitization induced by methylene blue has been well studied. A predominant type II photosensitizing activity, mediated by singlet oxygen, has been already demonstrated on various models. The purpose of this study is to compare this photosensitization system with that induced by naproxen, belonging to the class of non-steroidal anti-inflammatory drugs. For this compound, a type I (radical) and type II (singlet oxygen) cooperative mechanism of photoinduced damage was previously proposed. This study represents an example of testing drugs on the simple experimental model of amino acid residues in proteins. Particular emphasis is dedicated to modifications caused by the formation of drug photomediated toxic species, such as reactive oxygen species (ROS). This is achieved following the kinetics of photodegradation of the sensitizers and of the amino acid, as well as the formation of their photoproducts and by evaluation of quantum yields of the various processes. Tryptophan photoproducts represent biomarkers of oxidative damage indicative for protein photooxidation and for the molecular mechanism of photosensitization; some of these have been identified for the first time as UVA photosensitization products. The pattern of Trp photoproducts formed by the two compounds differs and is specific for each type of sensitization process. These observations support extending the investigation to systems of increasing molecular complexity, that is Trp in isolated proteins and in cells and represent an effort to provide a simplified rationale of the complex picture coming out from literature data and our experimental results.

First-principle conformational analysis of glycine residues in the αβ-tubulin dimer

Amino acids, especially glycine, have been extensively studied whereas their conformational behaviors in proteins are rather limited. In this work, all the polypeptides containing glycine residues are truncated from the alphabeta-tubulin dimer and refined with the partial optimization technique, where the backbone atoms of the previous and posterior residues to the glycine residues are fixed at the experimental Cartesian positions whereas the others fully relaxed. The combination of the polypeptide models and partial optimization technique is validated by twolayer ONIOM calculations, being effective to retain the local structures of proteins and meanwhile optimizing the concerned glycine residues towards energy minima. Owing to the lack of side chains, various types of hydrogen bonding interactions are detected in the glycine residues. The conformational analyses of proteins are mainly based on the dihedrals. Theta(1)( angleH(5)C(2)C(3)O(4)), Theta(2)( angleH(6)C(2)C(3)O(4)) and Theta(3)( angleN(1)C(2)C(3)O(4)) are the three key dihedrals to determine the glycine conformations, which were found to change synchronously and correlate with each other by four linear equations. It thus provided the first evidence of the correlation of intra-dihedrals for the amino acid residues in proteins. In addition, the three dihedrals of the gas-phase glycine conformations were also found to suit well with these linear equations, elucidating the rationality of using amino acids as the computational models of proteins.

Development of a derivatisation method for the analysis of aldehyde modified amino acid residues in proteins by Fourier transform mass spectrometry

A method was developed for the analysis of amino acids within bovine serum albumin (BSA) which had been modified by reaction with different enals. BSA was reacted with the aldehydes and the reaction products were stabilised by reaction with NaBH 4. The protein was then hydrolysed with 6N HCl and the hydrolysis products were analysed by liquid chromatography–mass spectrometry (LC–MS). The modified amino acids were derivatised with propylchloroformate. High resolution mass spectrometry carried out using an LTQ-Orbitrap instrument which was able to characterise a wide range of adducts. In addition double adducts were observed to be formed with 4-hydroxynonenal (HNE) and lysine or lysine + histidine. Qualitatively it was possible to consistently observe a pyridinium adduct formed between lysine and pentenal in human plasma from normal subjects.

Sequence-dependence and prediction of nucleotide solvent accessibility in double stranded DNA

Solvent accessibility of amino acid residues in proteins has been widely studied and many methods for its prediction from sequence and evolutionary information are available. Some of the advantages of studying amino acid solvent accessibility also apply to DNA. However, currently there are no methods to estimate the solvent accessibility of nucleotides, as most works on DNA structures have focused on elastic deformations and other structural attributes. In this work, an attempt has been made to analyze the distribution of different nucleotides in various accessibility ranges. Effect of neighboring nucleotides on the predictability of exposure has been evaluated by developing a linear perceptron model that takes sequence information as the input. Five different types of solvent accessibility (overall nucleotide, side chain, main chain, polar and non-polar) have been predicted. From the analysis, it is observed that Thymine stands out in terms of its higher exposed surface area, particularly its side chain and non-polar atoms. It is also concluded that the solvent accessibility of a nucleotide strongly depends on its sequence neighbors and can be predicted with fair success using this information.

Mass spectrometric analysis of HOCl- and free-radical-induced damage to lipids and proteins

In inflammatory diseases, release of oxidants leads to oxidative damage to biomolecules. HOCl (hypochlorous acid), released by the myeloperoxidase/H2O2/Cl- system, can cause formation of phospholipid chlorohydrins, or alpha-chloro-fatty aldehydes from plasmalogens. It can attack several amino acid residues in proteins, causing post-translational oxidative modifications of proteins, but the formation of 3-chlorotyrosine is one of the most stable markers of HOCl-induced damage. Soft-ionization MS has proved invaluable for detecting the occurrence of oxidative modifications to both phospholipids and proteins, and characterizing the products generated by HOCl-induced attack. For both phospholipids and proteins, the application of advanced mass spectrometric methods such as product or precursor ion scanning and neutral loss analysis can yield information both about the specific nature of the oxidative modification and the biomolecule modified. The ideal is to be able to apply these methods to complex biological or clinical samples, to determine the site-specific modifications of particular cellular components. This is important for understanding disease mechanisms and offers potential for development of novel biomarkers of inflammatory diseases. In the present paper, we review some of the progress that has been made towards this goal.

The impact of specific oxidized amino acids on protein turnover in J774 cells

Oxidized protein deposition and accumulation have been implicated in the aetiology of a wide variety of age-related pathologies. Protein oxidation in vivo commonly results in the in situ modification of amino acid side chains, generating new oxidized amino acid residues in proteins. We have demonstrated previously that certain oxidized amino acids can be (mis)incorporated into cell proteins in vitro via protein synthesis. In the present study, we show that incorporation of o- and m-tyrosine resulted in increased protein catabolism, whereas dopa incorporation generated proteins that were inefficiently degraded by cells. Incorporation of higher levels of L-dopa into proteins resulted in an increase in the activity of lysosomal cathepsins, increased autofluorescence and the generation of high-molecular-mass SDS-stable complexes, indicative of protein aggregation. These effects were due to proteins containing incorporated L-dopa, since they were not seen with the stereoisomer D-dopa, which enters the cell and generates the same reactive species as L-dopa, but cannot be incorporated into proteins. The present study highlights how the nature of the oxidative modification to the protein can determine the efficiency of its removal from the cell by proteolysis. Protection against the generation of dopa and other species that promote resistance to proteolysis might prove to be critical in preventing toxicity from oxidative stress in pathologies associated with protein deposition, such as atherosclerosis, Alzheimer's disease and Parkinson's disease.

InterProSurf: a web server for predicting interacting sites on protein surfaces

A new web server, InterProSurf, predicts interacting amino acid residues in proteins that are most likely to interact with other proteins, given the 3D structures of subunits of a protein complex. The prediction method is based on solvent accessible surface area of residues in the isolated subunits, a propensity scale for interface residues and a clustering algorithm to identify surface regions with residues of high interface propensities. Here we illustrate the application of InterProSurf to determine which areas of Bacillus anthracis toxins and measles virus hemagglutinin protein interact with their respective cell surface receptors. The computationally predicted regions overlap with those regions previously identified as interface regions by sequence analysis and mutagenesis experiments. The InterProSurf web server is available at http://curie.utmb.edu/

Liquid chromatographic analysis of phosphoamino acids at femtomole level using chemical derivatization with N-hydroxysuccinimidyl fluorescein- O-acetate

Phosphorylation of amino acid residues in proteins plays a major role in biological systems. In this paper, a reversed-phase high performance liquid chromatographic (HPLC) method based on chemical derivatization has been described for the separation and quantification of phosphoamino acids at femtomole level, using fluorimetric detection (FLD). The protocol involved pre-column derivatization of phosphoamino acids with N-hydroxysuccinimidyl fluorescein- O-acetate (SIFA) and subsequent separation on ZORBAX Eclipse XDB-C8 column. Several experimental factors that influenced derivatization and separation were carefully investigated. The derivatization was performed at 40 °C for 40 min in borate buffer (pH 8.5). Under the optimum conditions, phosphoserine (P-Ser), phosphothreonine (P-Thr) and phosphotyrosine (P-Tyr) were satisfactorily separated in 8 min. The detection limits (signal-to-noise ratio = 3) for the phosphoamino acids could reach 10–20 fmol, which was the lowest value reported for HPLC methods and comparable to those obtained by capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection methods. The proposed method has been validated and used to characterize the phosphoamino acids in the hydrolyzed phosphorylated protein samples. The results clearly demonstrated the potential of this technique to study phosphoamino acids as well as provided a new analytical methodology that should be applicable to the study of phosphorylation of protein in biological system.

SVM‐Cabins: Prediction of solvent accessibility using accumulation cutoff set and support vector machine

A number of methods for predicting levels of solvent accessibility or accessible surface area (ASA) of amino acid residues in proteins have been developed. These methods either predict regularly spaced states of relative solvent accessibility or an analogue real value indicating relative solvent accessibility. While discrete states of exposure can be easily obtained by post prediction assignment of thresholds to the predicted or computed real values of ASA, the reverse, that is, obtaining a real value from quantized states of predicted ASA, is not straightforward as a two-state prediction in such cases would give a large real valued errors. However, prediction of ASA into larger number of ASA states and then finding a corresponding scheme for real value prediction may be helpful in integrating the two approaches of ASA prediction. We report a novel method of obtaining numerical real values of solvent accessibility, using accumulation cutoff set and support vector machine. This so-called SVM-Cabins method first predicts discrete states of ASA of amino acid residues from their evolutionary profile and then maps the predicted states onto a real valued linear space by simple algebraic methods. Resulting performance of such a rigorous approach using 13-state ASA prediction is at least comparable with the best methods of ASA prediction reported so far. The mean absolute error in this method reaches the best performance of 15.1% on the tested data set of 502 proteins with a coefficient of correlation equal to 0.66. Since, the method starts with the prediction of discrete states of ASA and leads to real value predictions, performance of prediction in binary states and real values are simultaneously optimized.

Chemical toxicology: reactive intermediates and their role in pharmacology and toxicology

Reactive intermediates formed during the metabolism of drugs have been investigated extensively over the past decades. Today, interest in reactive intermediates in drug discovery is focused on minimising bioactivation in hopes of reducing the risk of causing so-called idiosyncratic toxicity. These efforts are justified based on the ‘hapten hypothesis’, namely, that on binding to protein, reactive intermediates may elicit an immune response to the modified protein, leading to a cascade of events that ultimately manifests as a toxic outcome. However, the pharmacological action of certain drugs depends on reactive intermediates that modify critical amino acid residues of proteins, typically enzymes, thereby altering their activity. Thus, the notion that reactive intermediates are inherently dangerous is unjustified. When a reactive intermediate is necessary for the desired pharmacological effect of a drug, the selectivity it displays towards the target protein is crucial, as off-target binding may produce unwanted toxicities. On the other hand, reactive intermediates may play no role in toxicity. This review provides a balanced perspective, primarily focusing on the proposed role of reactive intermediates in drug toxicity, while also highlighting examples in which they are involved in causing the desired pharmacology. It is hoped that this knowledge can help scientists involved in drug discovery and development in their challenging task of producing safe and effective drugs.

Structure of the genetic code suggested by the hydropathy correlation between anticodons and amino acid residues.

The correlation between hydropathies of anticodons and amino acids, detected by other authors utilizing scales of amino acid molecules in solution, was improved with the utilization of scales of amino acid residues in proteins. Three partitions were discerned in the correlation plot with the principal dinucleotides of anticodons (pDiN, excluding the wobble position). (a) The set of outliers of the correlation: Gly-CC, Pro-GG, Ser-GA and Ser-CU. The amino acids are consistently small, hydro-apathetic, stabilizers of protein N-ends, preferred in aperiodic protein conformations and belong to synthetases class II. The pDiN sequences are representative of the homogeneous sector (triplets NRR and NYY), distinguished from the mixed sector (triplets NRY and NYR), that depict a 70% correspondence to the synthetases class II and I, respectively. The triplet pairs proposed to be responsible for the coherence in the set of outliers are of the palindromic kind, where the lateral bases are the same, CCC: GGG and AGA: UCU. This suggests that UCU previously belonged to Ser, adding to other indications that the attribution of Arg to YCU was due to an expansion of the Arg-tRNA synthetase specificity. The other attributions produced two correlation sets. (b) One corresponds to the remaining pDiN of the homogeneous sector, containing both synthetase classes; its regression line overlapped the one formed by the remaining attributions to class II. (c) The other contains the pDiN of the mixed sector and produced steeper slopes, especially with the class I attributions. It is suggested that the correlation was established when the amino acid composition of the protein synthetases became progressively enriched and that the set of outliers were the earliest to have been fixed.

A two‐step method for the introduction of single or multiple defined point mutations into the genome of Saccharomyces cerevisiae

The introduction of defined mutations into open reading frames (ORF) or non-translated regions of the genome is important to study of the structure-function relationship of amino acid residues in proteins or that of sequence motifs at the genome level. We describe a simple two-step method for the introduction of defined single or multiple point mutations into the genome of Saccharomyces cerevisiae. This method circumvents the need for plasmid-based mutagenesis and thus ensures homogenous expression of the gene of interest within the cell population. It is based on the introduction of a selectable marker downstream of the gene of interest. This marker is then amplified with a gene-specific primer that harbours the desired point mutation, creating a selectable marker-tagged mutant version of the gene of interest. The mutant fragment is then integrated into the genome of a wild-type strain through homologous recombination. Successive rounds of amplification of the mutant loci with primers that introduce additional point mutations upstream of existing mutations will generate multiple defined mutations within a single gene. As a proof of principle, we have employed this method to generate a temperature-sensitive mutant version of the plasma membrane ATPase, pma1-7, which bears two point mutations (Pro434Ala and Gly789Ser). Phenotypic analysis of a pma1-7 haploid strain indicates that this allele has the same characteristics as the original pma1-7 allele. It confers a temperature-sensitive growth phenotype and the newly synthesized Pma1-7 protein is unstable and rapidly degraded.

Atom-wise statistics and prediction of solvent accessibility in proteins

In this work, we explore a novel method to broaden the scope of sequence-based predictions of solvent accessibility or accessible surface area (ASA) to the atomic level. All 167 heavy atoms from the 20 types of amino acid residues in proteins have been studied. An analysis of ASA distribution of these atomic groups in different proteins has been performed and rotamer-style libraries have been developed. We observe that the ASA of some atomic groups (e.g., backbone C and N atoms) can be estimated from the sequence environment within a mean absolute error of 2–3 Å 2. However, some side chain atoms such as CG in Pro, NH1 in Arg and NE2 in Gln show a strong variability making it more difficult to estimate their ASA from sequence environment. In general, the prediction of ASA becomes more difficult for atomic positions at the side chain extremities of long amino acid residues (aromatic side chain terminals being the exception). Several atomic groups are frequently exposed to solvent. Some of them have a bimodal distribution, suggesting two stable conformations in terms of their solvent exposure. More detailed understanding and prediction of solvent accessibility, i.e., at an atomic level is expected to help in bioinformatics approaches to structure prediction, functional relevance of atomic solvent accessibilities and other interaction analyses.

Products of Cu(II)-catalyzed oxidation of the N-terminal fragments of α-synuclein in the presence of hydrogen peroxide

Reactive oxygen species (ROS) may provide the covalent modifications of amino acid residues in proteins, formation of protein–protein cross-linkages, and oxidation of the protein backbone resulting in protein fragmentation. In an attempt to elucidate the products of the copper(II)-catalyzed oxidation of the (1–17), (1–28), (1–39) and (1–39)(A30P) fragments of α-synuclein, the high performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) methods and Cu(II) /hydrogen peroxide as a model oxidizing system were employed. Peptide solution (0.50 mM) was incubated at 37 °C for 24 h with metal:peptide:hydrogen peroxide molar ratio 1:1:4 in phosphate buffer, pH 7.4. Oxidation targets for all peptide studied are the methionine residues (M 1, M 5). Incubation 24 h of the (1–28), (1–39) and (1–39)(A30P) fragments in aerobic conditions lead to the oxidation of one methionine residue to methionine sulfoxide. Reaction of hydrogen peroxide with all fragments of α-synuclein resulted in oxidation of two methionine residues (M 1, M 5) to methionine sulfoxides. For the Cu(II):peptide:hydrogen peroxide 1:1:4 molar ratio systems the further oxidation of methionine residues to sulfone was observed. The cleavage of the peptide bond M 1–D 2 for all peptides studied was observed as metal binding residues. For the (1–39) and (1–39)(A30P) fragments of α-synuclein the molecular ions with lower molecular masses (A 11–Y 39, E 13–Y 39) were also detected.

Anomalous Orientation-Dependent Effective Pair Interaction among Histidine and Other Amino Acid Residues in Metalloproteins: Breakdown of the Hydropathy Scale Index

Hydropathy scale is widely used to obtain a measure of the effective interaction between any two amino acid residues in proteins and is based on the assumption that attraction between two hydrophobic groups and repulsion between hydrophilic groups (in water) can be translated straightforwardly to protein environment. Here we employ a recently developed statistical mechanical approach combined with the Protein Data Bank to obtain both distance- and orientation-dependent potential of mean force (ODPMF). This allowed us to explore effective pair potential among many amino acid residues and to examine the validity of the hydropathy scale in modeling the interaction among amino acid residues. We find that in some cases, like Phe-Phe and Lys-Lys, the hydropathy scale approach is largely obeyed. However, we also observe many unexpected pair interactions which defy the trend given by published hydropathy scales. An example of the former is the arginine-arginine (Arg-Arg) pair interaction which is found to be strongly and surprisingly attractive at short separation, even though it is the most hydrophilic residue. Here the head-to-head (see text) interaction is also stabilized. Tryptophan residues also exhibit strong attractive interaction. Equally important, we find strong influence of metal in determining effective interaction among the amino acid residues. It is the behavior of the histidine (His) which is found to be the most unusual. It exhibits a strong attractive interaction with itself which gets significantly enhanced in metalloproteins. These results highlight the important (sometime hidden) role of metals in protein structure and folding.