Structure-based identification and clustering of protein families and superfamilies

Summary We describe an approach to protein structure comparison designed to detect distantly related proteins of similar fold, where the procedure must be sufficiently flexible to take into account the elasticity of protein folds without losing specificity. Protein structures are represented as a series of secondary structure elements, where for each element a local environment describes its relations with the elements that surround it. Secondary structures are then aligned by comparing their features and local environments. The procedure is illustrated with searches of a database of 468 protein structures in order to identify proteins of similar topology to porcine pepsin, porphobilinogen deaminase and serum amyloid P-component. In all cases the searches correctly identify protein structures of similar fold as the search proteins. Multiple cross-comparisons of protein structures allow the clustering of proteins of similar fold. This is exemplified with a clustering of /- and -class protein structures. We discuss applications of the comparison and clustering of three-dimensional protein structures to comparative modelling and structure-based protein design.     

Ko-LABSUE: a quasi non-dependent data format package fork o-quantification

Thek _o-standardization method has been extensively used in the Pierre Süe laboratory during the last few years. We had developed two different new programs which both used parts of a commercial package (Canberra or Nuclear Data), for example: isotope parameter editor, automatic peak search, nuclide identification. However, further adaptation of these packages is not easy, due to the specific data handling of the two programs. In order to overcome these constraints, we decided to write a completely new program, Ko-LABSUE, which is as independent of the manufacturer specifications as possible. All of the different steps of thek _o-quantification (automatic peak search, nuclide identification, calculation of elemental concentration etc.) are processed by modules written in TURBO-PASCAL. Only two unit modules have to be re-written to process manufacturer data files of different formats: the modules used to read the spectrum data and the detector efficiency curve. This program runs on an IBM-PC and is currently on -test in our laboratory.     

PROGEN: An automated modelling algorithm for the generation of complete protein structures from the α-carbon atomic coordinates

Summary A modelling algorithm (PROGEN) for the generation of complete protein atomic coordinates from only the -carbon coordinates is described. PROGEN utilizes an optimal geometry parameter (OGP) database for the positioning of atoms for each amino acid of the polypeptide model. The OGP database was established by examining the statistical correlations between 23 different intra-peptide and inter-peptide geometric parameters relative to the -carbon distances for each amino acid in a library of 19 known proteins from the Brookhaven Protein Database (BPDB). The OGP files for specific amino acids and peptides were used to generate the atomic positions, with respect to -carbons, for main-chain and side-chain atoms in the modelled structure. Refinement of the initial model was accomplished using energy minimization (EM) and molecular dynamics techniques. PROGEN was tested using 60 known proteins in the BPDB, representing a wide spectrum of primary and secondary structures. Comparison between PROGEN models and BPDB crystal reference structures gave r.m.s.d. values for peptide main-chain atoms between 0.29 and 0.76 Å, with a grand average of 0.53 Å for all 60 models. The r.m.s.d. for all non-hydrogen atoms ranged between 1.44 and 1.93 Å for the 60 polypeptide models. PROGEN was also able to make the correct assignment of cis- or trans-proline configurations in the protein structures examined. PROGEN offers a fully automatic building and refinement procedure and requires no special or specific structural considerations for the protein to be modelled.     

A theoretical model for the Gla-TSR-EGF-1 region of the anticoagulant cofactor protein S: From biostructural pathology to specie

Protein S (PS), which functions as a species-specific anticoagulant cofactor to activated protein C (APC), is a mosaic protein that interacts with the phospholipid membrane via its -carboxyglutamate-rich (Gla) module. This module is followed by the thrombin-sensitive region (TSR), sensitive to thrombin cleavage, four epidermal growth factor (EGF)-like modules and a last region referred to as the sex hormone binding globulin (SHBG) domain. Of these, the TSR and the first EGF-like regions have been shown to be important for the species-specific interaction with APC. Difficulties in crystallising PS have so far hindered its study at the atomic level. Here, we report theoretical models for the Gla and EGF-1 modules of human PS constructed using prothrombin and factor X experimental structures. The TSR was built interactively. Analysis of the model linked with the large body of biochemical literature on PS and related proteins leads to suggestions that (i) the TSR stabilises the calcium-loaded Gla module through hydrophobic and ionic interactions and its conformation depends on the presence of the Gla module; (ii) the TSR does not form a calcium binding site but is protected from thrombin cleavage in the calcium-loaded form owing to short secondary structure elements and close contact with the Gla module; (iii) the PS missense mutations in this region are consistent with the structural data, except in one case which needs further investigation; and (iv) the two PS faces involving regions of residues Arg⁴⁹–Gln⁵²–Lys⁹⁷ (TSR-EGF-1) and Thr¹⁰³–Pro¹⁰⁶ (EGF-1) may be involved in species-specific interactions with APC as they are richer in nonconservative substitution when comparing human and bovine protein S. This preliminary model helps to plan future experiments and the resulting data will be used to further validate and optimise the present structure.     

Energetics of protein-cyclodextrin interactions

The energetics of interaction of a range of cyclodextrins with folded and unfolded proteins has been examined by sensitive microcalorimetry techniques. Weak interaction with exposed amino acid residues promotes unfolding and dissociation of proteins. The possibility that such interactions may facilitate the use of cyclodextrins as chaperone-mimics in the refolding of denatured protein has been explored with the enzyme phosphoglycerate kinase. Up to 40% regain of activity can be achieved in some cases.     

Fluorometric assay of amino acids and amines by use of 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole(NBD-F) in high-performance liquid chromatography

Summary The reaction of a newly developed fluoregenic reagent, 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole(NBD-F), with amino acids and biogenic amines was investigated. NBD-F was reactive to both primary and secondary amines including amino acids and biogenic amines such as catecholamines. The amino acids were reacted with the reagent, separated by high-performance liquid chromatography on -Bondapak C₁₈ and detected at 10 to 100 fmol level. A few g of protein hydrolysates, rabbit pyruvate kinase M₁, rabbit aldolase A and papain, were adequate for the amino acids quantitation. An automatic amino acid analyzer with fluorometric detection by the post-column derivatization with NBD-F enabled the amino acid profile analysis in blood samples present in a paper disc of 3 mm diameter.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Complex associations between membrane proteins analyzed by analytical ultracentrifugation: Studies on the erythrocyte membrane proteins band 3 and ankyrin

Associations between different water-soluble proteins can be studied by sedimentation equilibrium experiments in the analytical ultracentrifuge and subsequent mathematical analysis of thec(r)-distributions obtained. The analysis can be simplified by labelling one of the proteins with a dye absorbing at wavelengths >300 nm. The method can also be applied to intrinsic membrane proteins in solutions of a nonionic detergent. The present paper both reviews the method and reports application to the associations between two proteins of the human erythrocyte membrane: 1) band 3, the membrane's main intrinsic protein which, in detergent solutions and presumably also in the erythrocyte membrane, is in a monomer/dimer/tetramer association equilibrium, and 2) the cytoskeletal protein ankyrin which links the membrane skeleton to the lipid bilayer by binding to band 3. Ankyrin was labelled with fluorescein isothiocyanate and the detergent used was C₁₂E₉. It was found that the only aggregate of ankyrin and band 3 occurring in significant amounts was a complex of one ankyrin molecule and four band 3 molecules. This strongly suggests that, in the erythrocyte membrane, the band 3 tetramer represents the high affinity ankyrin binding site.This paper was presented at the VI. Symposium on Analytical Ultracentrifugation, Marburg, F.R.G., February 16–17, 1989.     

Generation of bright monomeric red fluorescent proteins via computational design of enhanced chromophore packing

Red fluorescent proteins (RFPs) have found widespread application in chemical and biological research due to their longer emission wavelengths. Here, we use computational protein design to increase the quantum yield and thereby brightness of a dim monomeric RFP (mRojoA, quantum yield = 0.02) by optimizing chromophore packing with aliphatic residues, which we hypothesized would reduce torsional motions causing non-radiative decay. Experimental characterization of the top 10 designed sequences yielded mSandy1 (λ_em = 609 nm, quantum yield = 0.26), a variant with equivalent brightness to mCherry, a widely used RFP. We next used directed evolution to further increase brightness, resulting in mSandy2 (λ_em = 606 nm, quantum yield = 0.35), the brightest Discosoma sp. derived monomeric RFP with an emission maximum above 600 nm reported to date. Crystallographic analysis of mSandy2 showed that the chromophore p-hydroxybenzylidene moiety is sandwiched between the side chains of Leu63 and Ile197, a structural motif that has not previously been observed in RFPs, and confirms that aliphatic packing leads to chromophore rigidification. Our results demonstrate that computational protein design can be used to generate bright monomeric RFPs, which can serve as templates for the evolution of novel far-red fluorescent proteins.

We used computational design to increase quantum yield in a fluorescent protein by optimizing chromophore packing to reduce non-radiative decay, resulting in an >10-fold increase in quantum yield that was further improved by directed evolution.     

A computational model of the nicotinic acetylcholine binding site

We have derived a model of the nicotinic acetylcholine binding site. This was accomplished by using three known agonists (acetylcholine, nicotine and epibatidine) as templates around which polypeptide side chains, found to be part of the receptor cavity from published molecular biology studies, are allowed to flow freely in molecular dynamics simulations and mold themselves around these templates. The resulting supramolecular complex should thus be a complement, both in terms of steric effects as well as electronic effects, to the agonists and it should be a good estimation of the true receptor cavity structure. The shapes of those minireceptor cavities equilibrated rapidly on the simulation time scale and their structural congruence is very high, implying that a satisfactory model of the nicotinic acetylcholine binding site has been achieved. The computational methodology was internally tested against two rigid and specific antagonists (dihydro--erytroidine and erysoidine), that are expected to give rise to a somewhat differently shaped binding site compared to that derived from the agonists. Using these antagonists as templates there were structural reorganizations of the initial receptor cavities leading to distinctly different cavities compared to agonists. This indicates that adequate times and temperatures were used in our computational protocols to achieve equilibrium structures for the agonists. Overall, both minireceptor geometries for agonists and antagonists are similar with the exception of one amino acid (ARG209).     

Enhancement of the chemiluminescence of permanganate-formaldehyde system by gold/silver nanoalloys and its application to trace determination of melamine

We have examined the specificity of electrosynthesized poly-o-phenylenediamine as a kind of material molecularly imprinted with metal chelates. Molecularly imprinted polymers (MIPs) were prepared in situ by an electrochemical method. The EDTA chelate complexes of Cu(II), Zn(II), Fe(III) and Cd(II) ions were used as templates of the MIPs deposited on a gold electrode of a quartz crystal microbalance (QCM). The binding ability and specificity of the MIPs were investigated via the frequency response of the QCM to the adsorption of the template chelates and their analogs. Linear relationships are observed between the frequency shift and the concentration of the template chelates over a typical range of ~10^?6 to ~10^?4?mol·L^?1. The results reveal good binding specificity of such MIPs for their templates over structurally related analogs, but the selectivity coefficients are less significant than that for enantiomers. The results also suggest no significant correlation between binding specificity and the ionic (or atomic) radius of the investigated metal ions. The observed specificity is qualitatively attributed to the overall conformational difference of the metal-EDTA complexes resulting from their difference in both ionic radius and electronic structures.

PPM-Dom: A novel method for domain position prediction

Domains are the structural basis of the physiological functions of proteins, and the prediction of which is an advantageous process on the study of protein structure and function. This article proposes a new complete automatic prediction method, PPM-Dom (Domain Position Prediction Method), for predicting the particular positions of domains in a target protein via its atomic coordinate. The presented method integrates complex networks, community division, and fuzzy mean operator (FMO). The whole sequences are divided into potential domain regions by the complex network and community division, and FMO allows the final determination for the domain position. This method will suffice to predict regions that will form a domain structure and those that are unstructured based on completely new atomic coordinate information of the query sequence, and be able to separate different domains in the same query sequence from each other. On evaluating the performance using an independent testing dataset, PPM-Dom reached 91.41% for prediction accuracy, 96.12% for sensitivity and 92.86% for specificity. The tool bag of PPM-Dom is freely available at http://cic.scu.edu.cn/bioinformatics/PPMDom.zip.     

Multiple Reaction Monitoring for Direct Quantitation of Intact Proteins Using a Triple Quadrupole Mass Spectrometer

Methods that can efficiently and effectively quantify proteins are needed to support increasing demand in many bioanalytical fields. Triple quadrupole mass spectrometry (QQQ-MS) is sensitive and specific, and it is routinely used to quantify small molecules. However, low resolution fragmentation-dependent MS detection can pose inherent difficulties for intact proteins. In this research, we investigated variables that affect protein and fragment ion signals to enable protein quantitation using QQQ-MS. Collision induced dissociation gas pressure and collision energy were found to be the most crucial variables for optimization. Multiple reaction monitoring (MRM) transitions for seven standard proteins, including lysozyme, ubiquitin, cytochrome c from both equine and bovine, lactalbumin, myoglobin, and prostate-specific antigen (PSA) were determined. Assuming the eventual goal of applying such methodology is to analyze protein in biological fluids, a liquid chromatography method was developed. Calibration curves of six standard proteins (excluding PSA) were obtained to show the feasibility of intact protein quantification using QQQ-MS. Linearity (2–3 orders), limits of detection (0.5–50 μg/mL), accuracy (<5% error), and precision (1%–12% CV) were determined for each model protein. Sensitivities for different proteins varied considerably. Biological fluids, including human urine, equine plasma, and bovine plasma were used to demonstrate the specificity of the approach. The purpose of this model study was to identify, study, and demonstrate the advantages and challenges for QQQ-MS-based intact protein quantitation, a largely underutilized approach to date.

Graphical Abstract

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Metrology for metalloproteins—where are we now, where are we heading?

The use of the amount of certain proteins in biological samples as markers for distinguishing between a healthy and a diseased state has become increasingly important in clinical diagnosis. As about 30 % of all proteins contain metals in one form or another, either as a cofactor or covalently bound as part of the protein, some of these proteins are regularly analyzed in clinical laboratories. With the increasing number of measurements of those proteins performed all over the world, the necessity of obtaining reliable and comparable results is becoming a focal point for scientists and politicians. Directives such as the EC directive covering in vitro diagnostic medical devices (Directive 98/79/EC) and standards such as EN ISO 17511:2003 demand the traceability of the results obtained for analytes in samples of human origin. However, no reference measurement procedures with results traceable to the SI exist for many metalloproteins. In this article, the situation for a few important metalloproteins, such as hemoglobin, transferrin, superoxide dismutase, ceruloplasmin, and C-reactive protein, for which specific efforts have been made in recent years to achieve comparable and traceable results worldwide, is discussed. These proteins also serve as examples of the difficulties scientists face when they wish to quantify proteins and the pitfalls they should avoid to achieve reliable results.

BUILDER v.2: Improving the chemistry of a de novo design strategy

Summary Significant improvements have been made to the de novo drug design program BUILDER. The BUILDER strategy is to find molecule templates that bind tightly to hot spots in the target receptor, and then generate bridges to join these templates. In this paper, the bridging algorithm has been further developed to improve the chemical sense and diversity of the bridges, as well as the robustness of the technique. The improved algorithm is then applied to rebuild known bridges in methotrexate and HIV protease. Finally, the entire BUILDER approach is tested by rebuilding methotrexate de novo.     

Histidine-specific bioconjugation via visible-light-promoted thioacetal activation

Histidine (His, H) undergoes various post-translational modifications (PTMs) and plays multiple roles in protein interactions and enzyme catalyzed reactions. However, compared with other amino acids such as Lys or Cys, His modification is much less explored. Herein we describe a novel visible-light-driven thioacetal activation reaction which enables facile modification on histidine residues. An efficient addition to histidine imidazole N3 under biocompatible conditions was achieved with an electrophilic thionium intermediate. This method allows chemo-selective modification on peptides and proteins with good conversions and efficient histidine-proteome profiling with cell lysates. 78 histidine containing proteins were for the first time found with significant enrichment, most functioning in metal accumulation in brain related diseases. This facile His modification method greatly expands the chemo-selective toolbox for histidine-targeted protein conjugation and helps to reveal histidine''s role in protein functions.

Functionalization of histidine residues in proteins via visible-light-promoted thioacetal activation is reported. ∼2000 proteins with reactive and exposed histidine residues from the MCF7 cell line are characterized using ABPP by this method.     

Introducing HYPERMET-PC for automatic analysis of complex gamma-ray spectra

In short time activation analysis prompt gamma-activation analysis and in high rate -ray spectroscopy in general, the shape parameters for peaks and back ground usually vary, rendering spectrum evaluation codes based on a fixed shape calibration unsuitable. An interactive version of the well-known, fully automatic -ray spectrum analysis code HYPERMET has been developed in C ++ for the IBM-PC. It runs under MS-DOS, in conventional memory, and can handle up to 16k-channel spectra, recorded with CANBERRA's System 100 and AccuSpec and with ORTEC's ACE plug-in MCA cards. A Windows-like graphics environment is provided with mouse controlled pull-down menus, pop-up windows and rubber band expansion. All basic features of HYPERMET such as fully automatic peak search, nonlinear fitting of multiplets with automatically adjusted Gaussian peak widths exponential tails and a complex background function have been retained. All details of the fitting procedure are recorded in a data base, hence any fitted region can be retrieved and modified interactively, even after a fully automatic spectrum evaluation. The program also provides an output peak list in SAMPO90 format for further processing. The latter format is widely used in a number of sample analysis programs such as KAYZERO a software package fork ₀ standardization in neutron activation analysis.     

Detection limits and precisions in various irradiation and counting regimes

The widespread introduction of rapid pneumatic sample transfer systems has enabled instrumental neutron activation analysis to be based on an increasing number of very short-lived activities. Furthermore, these transfer systems have been interfaced to computer-based MCA's so that the experimenter has complete control over irradiation, decay and counting times, as well as being able to arrange the automatic transfer of numbers of samples between the various stations. Thus the analyst now has a series of options available to him to make the best use of time and facilities. Based on the requirements of detection limits and precisions, he will choose between various irradiation and counting régimes (a) single i.e. conventional (b) cyclic and (c) repeated; or he may choose to replicate the sample a number of times. This paper examines how detection limits and precisions are affected by the above options. By considering a specific isotope, being detected in backgrounds of different half-lives, it is possible to calculate signal-to-noise ratios in each of these cases, and hence compare these régimes from this aspect. Based on calculations for the isotope^77mSe (17.5 s), which is now being widely accepted as the basis for selenium analysis, it is shown that, if a low detection limit is the prime consideration, then replicating samples is the procedure of choice; however, if commercial considerations of sample throughput are important then a pseudocyclic régime would provide the best compromise.     

The excited states of bipyridyl and phenanthroline complexes of Fe(III), Ru(II) and Ru(III): A molecular orbital study

The semiempirical zero-differential-overlap molecular orbital model which was shown in earlier papers in this series to give a good account of the charge transfer and -* spectra of Fe(II) complexes with conjugated ligands such as 2,2-bipyridyl and 1,10-phenanthroline is extended to complexes having openshell ground states, such as those of Fe(III), and to complexes of Ru(II) and Ru(III). The results are used to assign the observed charge transfer and intra-ligand absorption bands to specific orbital transitions. Observed and calculated intensities are in good agreement: reasons are advanced for the much lower intensity of the charge transfer bands in Ru(III) compared to Ru(II) complexes.     

Modeling of protease I collagenolytic enzyme from the fiddler crab Uca pugilator

Summary Collagenolytic protease I from the fiddler crab Uca pugilator belongs to the serine proteases of the trypsin family. A graphic molecular model was built using information from sequences and X-ray structures of four homologous proteins which were superimposed to define structurally conserved regions. Protease I sequence was aligned, with sequences of the model proteins, without permitting any deletion or insertion in these regions. Elastase -carbon chain was selected as a template molecule. For the structurally variable regions, fragments of the four homologous proteins which were closest in sequence were selected. Intramolecular steric hindrance, that resulted from the substitution of the residues of the templates by protease I residues, was corrected by adjustment of the side-chain conformational angles. The model was then optimized by energy minimization. The primary specificity pocket in the model of collagenolytic protease I predicts a substrate preference for both P1 hydrophobic and positively charged residues which is in agreement with the biochemical observations. As soybean trypsin inhibitor (STI) is known to inhibit collagenolytic protease I, a tentative model of the complex was constructed and possibilities of interaction examined.     

Water‐Soluble Methyl Orange Fibrils as Versatile Templates for the Fabrication of Conducting Polymer Microtubules

One‐dimensional methyl orange fibrils can be easily prepared. They are stable in acidic aqueous solutions and soluble in neutral water. When used to synthesize conducting polymer microtubules, the fibrils act as “hard templates” formally but as “soft templates” effectively. Microtubular structures of polypyrrole, polyaniline, and poly(3,4‐ethylenedioxythiophene) have been achieved successfully via such water‐soluble versatile templates.