Contemporary techniques for detecting and identifying proteins susceptible to reversible thiol oxidation

Elevated protein oxidation is a widely reported hallmark of most major diseases. Historically, this 'oxidative stress' has been considered causatively detrimental, as the protein oxidation events were interpreted simply as damage. However, recent advances have changed this antiquated view; sensitive methodology for detecting and identifying proteins susceptible to oxidation has revealed a fundamental role for this modification in physiological cell signalling during health. Reversible protein oxidation that is dynamically coupled with cellular reducing systems allows oxidative protein modifications to regulate protein function, analogous to phosphoregulation. However, the relatively labile nature of many reversible protein oxidation states hampers the reliable detection and identification of modified proteins. Consequently, specialized methods to stabilize protein oxidation in combination with techniques to detect specific types of modification have been developed. Here, these techniques are discussed, and their sensitivity, selectivity and ability to reliably identify reversibly oxidized proteins are critically assessed.     

Hydrophobicity and structural classes in proteins.

The bulk hydrophobic character for the 20 natural amino acid residues, has been obtained from a database of 60 protein structures, grouped in the four structural classes alpha alpha, beta beta, alpha + beta and alpha/beta. The hydrophobicity coefficients thus obtained are compared with Ponnuswamy's original values using scales normalized to average = 0.0 and standard deviation = 1.0. Even though most of the amino acid residues do not change their hydropathic character in the different structural classes, their behaviour suggests the convenience that averaging methods should only consider proteins of the same structural class and that this information should be included in the secondary structure methods.     

Hydrophobicity and residue-residue contacts in globular proteins

A comprehensive statistical analysis of residue-residue contacts and residue environment in protein 3-D structures is presented. In the present work the range of interresidue interactions (effective radius of influence) in tertiary structures of proteins is examined and found to be 10 Å. This result is obtained by correlating the average number of residues within a spherical volume of different radii (contact numbers) with hydrophobicity. Best correlations are obtained with a radius of 10 Å. The same result is obtained when (i) only long-range interactions are considered and (ii) representative side chain atoms are used to indicate the tertiary structure instead of the usual representation of C^α atoms. Residue environment has been investigated using similar methods. Environmental hydrophobicity varies within only a small range of all residue types. Other physicochemical properties also exhibit similar trends of variation, and only five hydrophobic residues (Leu, Val, Met, Phe and Ile) produce a decrement of around 10% from the expected mean of the physicochemical distance between a residue type and its average environment. An information theory approach is proposed to compare domains, which takes into account the effective radius of influence of residues and sequence similarity.     

Hydrophobicity and stability for a family of model proteins

According to the conventional definition, the hydrophobic effect is a result of thermodynamic changes occurring when a nonpolar group dissolves in water and attributable to the fact that water in contact with such a group has special structural and energetic properties. Disagreement now exists as to whether this effect promotes or hinders protein denaturation. Taking the heat capacity change of unfolding as a measure of the hydrophobicity of the protein interior, others have shown that protein stabilities are systematically affected by changes in hydrophobicity. It has been suggested that the observed trends show that hydrophobic hydration is intrinsically a destabilizing factor. Model calculations using known equations for the stability curves and certain simplifying assumptions now show that such regularities provide no evidence for or against this conclusion. All available data can be rationalized if hydrophobic terms are evaluated from models that require a positive hydrophobic contribution to the Gibbs energy of unfolding. The calculations also confirm the recent finding that any set of proteins with denaturation temperatures between about 330 and 380 K that exhibits entropy convergence at about 386 K is thermodynamically required to show enthalpy convergence at approximately the same temperature. © 1993 John Wiley & Sons, Inc.     

A method of searching for amphipathic structures in protein sequences

A simple method for searching amphipathic helices based on estimation of correlation between hydrophobicity distribution and periodic function is proposed. The method was examined in a series of proteins with known T-cell epitopes, which are mostly amphipathic helices. The predictive power of the method is discussed.     

Hydrophobicity of amino acid subgroups in proteins

Protein folding studies often utilize areas and volumes to assess the hydrophobic contribution to conformational free energy (Richards, F.M. Annu. Rev. Biophys. Bioeng. 6:151-176, 1977). We have calculated the mean area buried upon folding for every chemical group in each residue within a set of X-ray elucidated proteins. These measurements, together with a standard state cavity size for each group, are documented in a table. It is observed that, on average, each type of group buries a constant fraction of its standard state area. The mean area buried by most, though not all, groups can be closely approximated by summing contributions from three characteristic parameters corresponding to three atom types: (1) carbon or sulfur, which turn out to be 86% buried, on average; (2) neutral oxygen or nitrogen, which are 40% buried, on average; and (3) charged oxygen or nitrogen, which are 32% buried, on average.     

Analyzing the structures, functions and evolution of two abundant gastrointestinal fatty acid binding proteins with recombinant DNA and computational techniques

The structures of intestinal and liver fatty acid binding proteins (FABPs) have been determined from an analysis of the nucleotide sequences of cloned cDNAs. The primary translation product of intestinal FABP mRNA contains 132 residues (Mr = 15 124). Liver FABP mRNA encodes a 127 amino acid polypeptide (Mr = 14 273). In vitro co-translational cleavage and translocation assays showed that neither sequence has a cleavable signal peptide or signal peptide equivalent - suggesting that the FABPs do not enter the secretory apparatus but rather are targeted to the cytoplasm. A variety of computational techniques were used to compare the two FABP sequences. The results indicate that liver and intestinal FABP are paralogous homologues. A superfamily of proteins was defined which includes the FABPs, the cellular retinol and retinoic acid binding proteins, the P2 protein of peripheral nerve myelin, and a polypeptide known as 422 whose synthesis is induced during differentiation of 3T3-L1 cells to adipocytes. No sequence homologies were noted between any of these small molecular weight cytosolic proteins and nonspecific lipid transfer protein (sterol carrier protein 2), phosphatidylcholine transfer protein, serum albumin or apolipoprotein AI. The FABPs may have structural features responsible for lipid-protein interactions that are not present in these non-homologous sequences. The distribution of intestinal and liver FABP mRNAs in adult rat tissues and the changes in FABP gene expression which occur during gastrointestinal development support the notion that these proteins are involved in fatty acid uptake, transport and/or compartmentalization. However, differences in tissue distribution and periods of non-coordinate expression during gastrointestinal ontogeny suggest that the two FABPs have distinct functions. The relationship between intestinal and liver FABPs and similar sized cytosolic FABPs isolated from brain, skeletal and cardiac muscle remains unclear. Recombinant DNA techniques combined with comparative sequence analyses offer a useful approach for defining unique as well as general structure-function relationships in this group of fatty acid binding proteins.     

Hydrophobicity scale for proteins based on inverse temperature transitions.

In general, proteins fold with hydrophobic residues buried, away from water. Reversible protein folding due to hydrophobic interactions results from inverse temperature transitions where folding occurs on raising the temperature. Because homoiothermic animals constitute an infinite heat reservoir, it is the transition temperature, Tt, not the endothermic heat of the transition, that determines the hydrophobically folded state of polypeptides at body temperature. Reported here is a new hydrophobicity scale based on the values of Tt for each amino acid residue as a guest in a natural repeating peptide sequence, the high polymers of which exhibit reversible inverse temperature transitions. Significantly, a number of ways have been demonstrated for changing Tt such that reversibly lowering Tt from above to below physiological temperature becomes a means of isothermally and reversibly driving hydrophobic folding. Accordingly, controlling Tt becomes a mechanism whereby proteins can be induced to carry out isothermal free energy transduction.     

Mathematical and computational modeling in biology at multiple scales

A variety of topics are reviewed in the area of mathematical and computational modeling in biology, covering the range of scales from populations of organisms to electrons in atoms. The use of maximum entropy as an inference tool in the fields of biology and drug discovery is discussed. Mathematical and computational methods and models in the areas of epidemiology, cell physiology and cancer are surveyed. The technique of molecular dynamics is covered, with special attention to force fields for protein simulations and methods for the calculation of solvation free energies. The utility of quantum mechanical methods in biophysical and biochemical modeling is explored. The field of computational enzymology is examined.     

Algorithms for computational solvent mapping of proteins

Computational mapping methods place molecular probes (small molecules or functional groups) on a protein surface to identify the most favorable binding positions by calculating an interaction potential. We have developed a novel computational mapping program called CS-Map (computational solvent mapping of proteins), which differs from earlier mapping methods in three respects: (i) it initially moves the ligands on the protein surface toward regions with favorable electrostatics and desolvation, (ii) the final scoring potential accounts for desolvation, and (iii) the docked ligand positions are clustered, and the clusters are ranked on the basis of their average free energies. To understand the relative importance of these factors, we developed alternative algorithms that use the DOCK and GRAMM programs for the initial search. Because of the availability of experimental solvent mapping data, lysozyme and thermolysin are considered as test proteins. Both DOCK and GRAMM speed up the initial search, and the combined algorithms yield acceptable mapping results. However, the DOCK-based approaches place the consensus site farther from its experimentally determined position than CS-Map, primarily because of the lack of a solvation term in the initial search. The GRAMM-based program also finds the correct consensus site for thermolysin. We conclude that good sampling is the most important requirement for successful mapping, but accounting for desolvation and clustering of ligand positions also help to reduce the number of false positives.     

Comparison of NMR and crystal structures of membrane proteins and computational refinement to improve model quality

Membrane proteins are challenging to study and restraints for structure determination are typically sparse or of low resolution because the membrane environment that surrounds them leads to a variety of experimental challenges. When membrane protein structures are determined by different techniques in different environments, a natural question is “which structure is most biologically relevant?” Towards answering this question, we compiled a dataset of membrane proteins with known structures determined by both solution NMR and X‐ray crystallography. By investigating differences between the structures, we found that RMSDs between crystal and NMR structures are below 5 Å in the membrane region, NMR ensembles have a higher convergence in the membrane region, crystal structures typically have a straighter transmembrane region, have higher stereo‐chemical correctness, and are more tightly packed. After quantifying these differences, we used high‐resolution refinement of the NMR structures to mitigate them, which paves the way for identifying and improving the structural quality of membrane proteins.     

Cell culture-based biosensing techniques for detecting toxicity in water

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Screening techniques for detecting allelic variation in DNA sequences

This article reviews four 'DNA screening techniques', namely heteroduplex analysis, single-strand conformational polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) as tools for the study of allelic variation in natural populations. The resolving power, advantages, and limitations of each technique are discussed and compared. We also provide some criteria for choosing among techniques and illustrate some practical issues with examples taken primarily from our own laboratory experience.     

Analytical and computational techniques for exogenous depolymerization of xenobiotic polymers

We analyze a mathematical model for exogenous depolymerization of xenobiotic polymers. We derive a sufficient condition for the solvability of its inverse problem to determine degradation rates. We also introduce a numerical technique to solve the inverse problem with an example to show how our numerical technique is applied to gel permeation chromatography data of polyethylene wax obtained before and after three-week cultivation of a fungus Aspergillus sp. AK-3. In addition, we present a numerical result which we obtained by applying the degradation rate based on the three week cultivation to simulate the transition of the weight distribution after cultivation of the fungus for five weeks.     

Molecular beacons for detecting DNA binding proteins

We report here a simple, rapid, homogeneous fluorescence assay, the molecular beacon assay, for the detection and quantification of sequence-specific DNA-binding proteins. The central feature of the assay is the protein-dependent association of two DNA fragments each containing about half of a DNA sequence defining a protein-binding site. Protein-dependent association of DNA fragments can be detected by any proximity-based spectroscopic signal, such as fluorescence resonance energy transfer (FRET) between fluorochromes introduced into these DNA molecules. The assay is fully homogeneous and requires no manipulations aside from mixing of the sample and the test solution. It offers flexibility with respect to the mode of signal detection and the fluorescence probe, and is compatible with multicolor simultaneous detection of several proteins. The assay can be used in research and medical diagnosis and for high-throughput screening of drugs targeted to DNA-binding proteins.     

Prion-like proteins and their computational identification in proteomes

Introduction: The aberrant or misfolded forms of the prion protein have been described as the causative agents of rare transmissible spongiform encephalopathies. In addition, proteins associated with frequently occurring neurodegenerative disorders, such as Alzheimer’s and Parkinson’s, are shown to share prion-like properties and to spread the disease in the brain.

Areas covered: Interest in the prion phenomenon has crystallized in a series of computational methods aimed at uncovering prion-like proteins at the proteome level. These programs rely on the identification of sequence signatures similar to those of yeast prions, whose structural conversion is driven by specific domains enriched in glutamine/asparagine residues. A myriad of prion-like candidates, similar to those in yeast, are predicted to exist in organisms across all kingdoms of life. We review here the role of prions, prionoids and prion-like proteins in health and disease, with a special focus on the algorithms and databases developed for their prediction and classification.

Expert commentary: Computational approaches provide novel insights into prion-like protein functions, their regulation and their role in disease.     

A procedure for detecting structural domains in proteins.

A procedure is described for detecting domains in proteins of known structure. The method is based on the intuitively simple idea that each domain should contain an identifiable hydrophobic core. By applying the algorithm described in the companion paper (Swindells MB, 1995, Protein Sci 4:93-102) to identify distinct cores in multi-domain proteins, one can use this information to determine both the number and the location of the constituent domains. Tests have shown the procedure to be effective on a number of examples, even when the domains are discontinuous along the sequence. However, deficiencies also occur when hydrophobic cores from different domains continue through the interface region and join one another.