Search PubMed⌕ Search

Biomedical subjects

Thomas Huber

Publications and source records attributed to Thomas Huber.

At least 19 recordsLinked to original sources
PubMed2006

Focusing in on structural genomics: the University of Queensland structural biology pipeline.

The flood of new genomic sequence information together with technological innovations in protein structure determination have led to worldwide structural genomics (SG) initiatives. The goals of SG initiatives are to accelerate the process of protein structure determination, to fill in protein fold space and to provide information about the function of uncharacterized proteins. In the long-term, these outcomes are likely to impact on medical biotechnology and drug discovery, leading to a better understanding of disease as well as the development of new therapeutics. Here we describe the high throughput pipeline established at the University of Queensland in Australia. In this focused pipeline, the targets for structure determination are proteins that are expressed in mouse macrophage cells and that are inferred to have a role in innate immunity. The aim is to characterize the molecular structure and the biochemical and cellular function of these targets by using a parallel processing pipeline. The pipeline is designed to work with tens to hundreds of target gene products and comprises target selection, cloning, expression, purification, crystallization and structure determination. The structures from this pipeline will provide insights into the function of previously uncharacterized macrophage proteins and could lead to the validation of new drug targets for chronic obstructive pulmonary disease and arthritis.

Animals↗
PubMed2006

Curvature and hydrophobic forces drive oligomerization and modulate activity of rhodopsin in membranes.

G protein-coupled receptors (GPCRs) are essential components of cellular signaling pathways. They are the targets of many current pharmaceuticals and are postulated to dimerize or oligomerize in cellular membranes in conjunction with their functional mechanisms. We demonstrate using fluorescence resonance energy transfer how association of rhodopsin occurs by long-range lipid-protein interactions due to geometrical forces, yielding greater receptor crowding. Constitutive association of rhodopsin is promoted by a reduction in membrane thickness (hydrophobic mismatch), but also by an increase in protein/lipid molar ratio, showing the importance of interactions extending well beyond a single annulus of boundary lipids. The fluorescence data correlate with the pK(a) for the MI-to-MII transition of rhodopsin, where deprotonation of the retinylidene Schiff base occurs in conjunction with helical movements leading to activation of the photoreceptor. A more dispersed membrane environment optimizes formation of the MII conformation that results in visual function. A flexible surface model explains both the dispersal and activation of rhodopsin in terms of bilayer curvature deformation (strain) and hydrophobic solvation energy. The bilayer stress is related to the lateral pressure profile in terms of the spontaneous curvature and associated bending rigidity. Transduction of the strain energy (frustration) of the bilayer drives protein oligomerization and conformational changes in a coupled manner. Our findings illuminate the physical principles of membrane protein association due to chemically nonspecific interactions in fluid lipid bilayers. Moreover, they yield a conceptual framework for understanding how the tightly regulated lipid compositions of cellular membranes influence their protein-mediated functions.

Animals↗
PubMed2006

Efficient chi-tensor determination and NH assignment of paramagnetic proteins.

Anisotropic magnetic susceptibility tensors chi of paramagnetic metal ions are manifested in pseudocontact shifts, residual dipolar couplings, and other paramagnetic observables that present valuable long-range information for structure determinations of protein-ligand complexes. A program was developed for automatic determination of the chi-tensor anisotropy parameters and amide resonance assignments in proteins labeled with paramagnetic metal ions. The program requires knowledge of the three-dimensional structure of the protein, the backbone resonance assignments of the diamagnetic protein, and a pair of 2D 15N-HSQC or 3D HNCO spectra recorded with and without paramagnetic metal ion. It allows the determination of reliable chi-tensor anisotropy parameters from 2D spectra of uniformly 15N-labeled proteins of fairly high molecular weight. Examples are shown for the 185-residue N-terminal domain of the subunit epsilon from E. coli DNA polymerase III in complex with the subunit theta and La3+ in its diamagnetic and Dy3+, Tb3+, and Er3+ in its paramagnetic form.

Algorithms↗
PubMed2006

Prediction of cis/trans isomerization in proteins using PSI-BLAST profiles and secondary structure information.

BACKGROUND: The majority of peptide bonds in proteins are found to occur in the trans conformation. However, for proline residues, a considerable fraction of Prolyl peptide bonds adopt the cis form. Proline cis/trans isomerization is known to play a critical role in protein folding, splicing, cell signaling and transmembrane active transport. Accurate prediction of proline cis/trans isomerization in proteins would have many important applications towards the understanding of protein structure and function. RESULTS: In this paper, we propose a new approach to predict the proline cis/trans isomerization in proteins using support vector machine (SVM). The preliminary results indicated that using Radial Basis Function (RBF) kernels could lead to better prediction performance than that of polynomial and linear kernel functions. We used single sequence information of different local window sizes, amino acid compositions of different local sequences, multiple sequence alignment obtained from PSI-BLAST and the secondary structure information predicted by PSIPRED. We explored these different sequence encoding schemes in order to investigate their effects on the prediction performance. The training and testing of this approach was performed on a newly enlarged dataset of 2424 non-homologous proteins determined by X-Ray diffraction method using 5-fold cross-validation. Selecting the window size 11 provided the best performance for determining the proline cis/trans isomerization based on the single amino acid sequence. It was found that using multiple sequence alignments in the form of PSI-BLAST profiles could significantly improve the prediction performance, the prediction accuracy increased from 62.8% with single sequence to 69.8% and Matthews Correlation Coefficient (MCC) improved from 0.26 with single local sequence to 0.40. Furthermore, if coupled with the predicted secondary structure information by PSIPRED, our method yielded a prediction accuracy of 71.5% and MCC of 0.43, 9% and 0.17 higher than the accuracy achieved based on the singe sequence information, respectively. CONCLUSION: A new method has been developed to predict the proline cis/trans isomerization in proteins based on support vector machine, which used the single amino acid sequence with different local window sizes, the amino acid compositions of local sequence flanking centered proline residues, the position-specific scoring matrices (PSSMs) extracted by PSI-BLAST and the predicted secondary structures generated by PSIPRED. The successful application of SVM approach in this study reinforced that SVM is a powerful tool in predicting proline cis/trans isomerization in proteins and biological sequence analysis.

Databases, Protein↗
PubMed2006

Site-specific labelling of proteins with a rigid lanthanide-binding tag.

This paper describes a generic method for the site-specific attachment of lanthanide complexes to proteins through a disulfide bond. The method is demonstrated by the attachment of a lanthanide-binding peptide tag to the single cysteine residue present in the N-terminal DNA-binding domain of the Escherichia coli arginine repressor. Complexes with Y(3+), Tb(3+), Dy(3+), Ho(3+), Er(3+), Tm(3+) and Yb(3+) ions were formed and analysed by NMR spectroscopy. Large pseudocontact shifts and residual dipolar couplings were induced by the lanthanide-binding tag in the protein NMR spectrum, a result indicating that the tag was rigidly attached to the protein. The axial components of the magnetic susceptibility anisotropy tensors determined for the different lanthanide ions were similarly but not identically oriented. A single tag with a single protein attachment site can provide different pseudocontact shifts from different magnetic susceptibility tensors and thus provide valuable nondegenerate long-range structure information in the determination of 3D protein structures by NMR spectroscopy.

Amino Acid Sequence↗
PubMed2006

Molecular insights into the self-assembly mechanism of dystrophia myotonica kinase.

Self-assembly via coiled-coil domains (CC) is crucial for the regulation of the dystrophia myotonica kinase (DMPK) -related family of kinases. These CC domains are thought to form dimeric arrangements and thus to mediate dimerization in these enzymes. Using size exclusion chromatography combined with multiangle static light scattering, we analyzed the oligomeric state of DMPK as well as that of a truncated variant lacking the CC fraction. Remarkably, both forms were found to assemble into robust dimers. In contrast, the CC domain in isolation yielded trimeric assemblies, indicating that the oligomerization properties of CC domains from this kinase family are more diversified than anticipated. The crystal structure of this CC has been elucidated to 1.6 angstroms resolution and its properties in solution established using sedimentation equilibrium and thermal denaturation. These data show that, contrary to expectations, the self-assembly of DMPK is not dictated by the association properties of its CC domain. Instead, it appears to be driven by sequence segments flanking both N and C termini of the catalytic kinase fraction, as suggested by models of head-to-head dimers based on small angle X-ray scattering data. Our findings support a shared pattern of assembly across DMPK, ROCKs, and MRCK members of this family.

Animals↗
PubMed2005

The Hiroshima thermal-neutron discrepancy for (36)Cl at large distances. Part I: New (36)Cl measurements in granite samples exposed to A-bomb neutrons.

The long-lived radioisotope (36)Cl (half-life: 301,000 years) was measured in granite samples exposed to A-bomb neutrons at distances from 94 to 1,591 m from the hypocenter in Hiroshima, by means of accelerator mass spectrometry (AMS). Measured (36)Cl/Cl ratios decrease from 1.6 x 10(-10) close to the hypocenter to about 1-2 x 10(-13), at a distance of 1,300 m from the hypocenter. At this distance and beyond the measured (36)Cl/Cl ratios do not change significantly and scatter around values of 1-2 x 10(-13). These findings suggest that the (36)Cl had been predominantly produced by thermalized neutrons from the A-bomb via neutron capture on stable (35)Cl, at distances from the hypocenter smaller than about 1,200 m. At larger distances, however, confounding processes induced by cosmic rays or neutrons from the decay of uranium and thorium become important. This hypothesis is theoretically and experimentally supported in a consecutive paper. The results are compared to calculations that are based on the most recent dosimetry system DS02. Close to the hypocenter, measured (36)Cl/Cl ratios are lower than those calculated, while they are significantly higher at large distances from the hypocenter. If the contribution of the cosmic rays and of the neutrons from the decay of uranium and thorium in the sample was subtracted, however, no significant deviation from the DS02 calculations was observed, at those distances. Thus, the Hiroshima neutron discrepancy reported in the literature for (36)Cl for samples from large distances from the hypocenter, i.e., higher measured (36)Cl/Cl ratios than predicted by the previous dosimetry system DS86, was not confirmed.

Biophysical Phenomena↗
PubMed2005

The Hiroshima thermal-neutron discrepancy for (36)Cl at large distances. Part II: Natural in situ production as a source.

For Hiroshima, a large discrepancy between calculated and measured thermal-neutron fluences had been reported in the past, for distances to the epicenter larger than about 1,000 m. To be more specific, measured (36)Cl concentrations in environmental samples from Hiroshima were too large at these distances, and the ratio of measured to calculated values reached about 70, at a distance of 1,800 m. In an attempt to identify other sources that might also produce (36)Cl in Hiroshima samples, the role of cosmic rays and of neutrons from natural terrestrial sources was investigated. Four reaction mechanisms were taken into account: spallation reactions of the nucleonic (hadronic) component of the cosmic rays on potassium (K) and calcium (Ca) in the sample material, particle emission after nuclear capture of negative muons by K and Ca, reactions of fast-muon induced electromagnetic, and hadronic showers with K and Ca, and neutron capture reactions with (35)Cl in the sample where the neutrons originate from the above three reaction mechanisms and from uranium and thorium decay. These mechanisms are physically described and mathematically quantified. It is shown that among those parameters important for the production of (36)Cl in granite, the chemical composition of the sample, the depth in the quarry where the sample had initially been taken, and the erosion rate at the site of the quarry are most important. Based on these physical, chemical, and geological parameters, (36)Cl concentrations were calculated for different types of granite that are typical for the Hiroshima area. In samples that were of these granite types and that had not been exposed to atomic bomb(A-bomb) neutrons, the (36)Cl concentration was also determined experimentally by means of accelerator mass spectrometry, and good agreement was found with the calculated values. The (36)Cl signal due to natural in situ production was also calculated in granite samples that had been exposed to A-bomb neutrons at distances up to 1,500 m from the hypocenter. It is demonstrated that, for granite samples from Hiroshima exposed to A-bomb neutrons beyond distances of about 1,300 m from the hypocenter, the (36)Cl signal is dominated by natural in situ production.

Biophysical Phenomena↗
PubMed2005

Modelling the structure of latexin-carboxypeptidase A complex based on chemical cross-linking and molecular docking.

We have determined the three-dimensional structure of the protein complex between latexin and carboxypeptidase A using a combination of chemical cross-linking, mass spectrometry and molecular docking. The locations of three intermolecular cross-links were identified using mass spectrometry and these constraints were used in combination with a speed-optimised docking algorithm allowing us to evaluate more than 3 x 10(11) possible conformations. While cross-links represent only limited structural constraints, the combination of only three experimental cross-links with very basic molecular docking was sufficient to determine the complex structure. The crystal structure of the complex between latexin and carboxypeptidase A4 determined recently allowed us to assess the success of this structure determination approach. Our structure was shown to be within 4 A r.m.s. deviation of Calpha atoms of the crystal structure. The study demonstrates that cross-linking in combination with mass spectrometry can lead to efficient and accurate structural modelling of protein complexes.

Algorithms↗
PubMed2005

Pilot studies on the parallel production of soluble mouse proteins in a bacterial expression system.

We investigated the parallel production in medium throughput of mouse proteins, using protocols that involved recombinatorial cloning, protein expression screening and batch purification. The methods were scaled up to allow the simultaneous processing of tens or hundreds of protein samples. Scale-up was achieved in two stages. In an initial study, 30 targets were processed manually but with common protocols for all targets. In the second study, these protocols were applied to 96 target proteins that were processed in an automated manner. The success rates at each stage of the study were similar for both the manual and automated approaches. Overall, 15 of the selected 126 target mouse genes (12%) yielded soluble protein products in a bacterial expression system. This success rate compares favourably with other protein screening projects, particularly for eukaryotic proteins, and could be further improved by modifications at the cloning step.

Animals↗
PubMed2005

An inflammatory role for the mammalian carboxypeptidase inhibitor latexin: relationship to cystatins and the tumor suppressor TIG1.

Latexin, the only known mammalian carboxypeptidase inhibitor, has no detectable sequence similarity with plant and parasite inhibitors, but it is related to a human putative tumor suppressor protein, TIG1. Latexin is expressed in the developing brain, and we find that it plays a role in inflammation, as it is expressed at high levels and is inducible in macrophages in concert with other protease inhibitors and potential protease targets. The crystal structure of mouse latexin, solved at 1.83 A resolution, shows no structural relationship with other carboxypeptidase inhibitors. Furthermore, despite a lack of detectable sequence duplication, the structure incorporates two topologically analogous domains related by pseudo two-fold symmetry. Surprisingly, these domains share a cystatin fold architecture found in proteins that inhibit cysteine proteases, suggesting an evolutionary and possibly functional relationship. The structure of the tumor suppressor protein TIG1 was modeled, revealing its putative membrane binding surface.

Amino Acid Motifs↗
PubMed2005

Translational incorporation of L-3,4-dihydroxyphenylalanine into proteins.

An Escherichia coli cell-free transcription/translation system was used to explore the high-level incorporation of L-3,4-dihydroxyphenylalanine (DOPA) into proteins by replacing tyrosine with DOPA in the reaction mixtures. ESI-MS showed specific incorporation of DOPA in place of tyrosine. More than 90% DOPA incorporation at each tyrosine site was achieved, allowing the recording of clean 15N-HSQC NMR spectra. A redox-staining method specific for DOPA was shown to provide a sensitive and generally applicable method for assessing the cell-free production of proteins. Of four proteins produced in soluble form in the presence of tyrosine, two resulted in insoluble aggregates in the presence of high levels of DOPA. DOPA has been found in human proteins, often in association with various disease states that implicate protein aggregation and/or misfolding. Our results suggest that misfolded and aggregated proteins may result, in principle, from ribosome-mediated misincorporation of intracellular DOPA accumulated due to oxidative stress. High-yield cell-free protein expression systems are uniquely suited to obtain rapid information on solubility and aggregation of nascent polypeptide chains.

Amino Acid Sequence↗
PubMed2004

Protein contact prediction using patterns of correlation.

We describe a new method for using neural networks to predict residue contact pairs in a protein. The main inputs to the neural network are a set of 25 measures of correlated mutation between all pairs of residues in two "windows" of size 5 centered on the residues of interest. While the individual pair-wise correlations are a relatively weak predictor of contact, by training the network on windows of correlation the accuracy of prediction is significantly improved. The neural network is trained on a set of 100 proteins and then tested on a disjoint set of 1033 proteins of known structure. An average predictive accuracy of 21.7% is obtained taking the best L/2 predictions for each protein, where L is the sequence length. Taking the best L/10 predictions gives an average accuracy of 30.7%. The predictor is also tested on a set of 59 proteins from the CASP5 experiment. The accuracy is found to be relatively consistent across different sequence lengths, but to vary widely according to the secondary structure. Predictive accuracy is also found to improve by using multiple sequence alignments containing many sequences to calculate the correlations.

Amino Acids↗
PubMed2004

Wurst: a protein threading server with a structural scoring function, sequence profiles and optimized substitution matrices.

Wurst is a protein threading program with an emphasis on high quality sequence to structure alignments (http://www.zbh.uni-hamburg.de/wurst). Submitted sequences are aligned to each of about 3000 templates with a conventional dynamic programming algorithm, but using a score function with sophisticated structure and sequence terms. The structure terms are a log-odds probability of sequence to structure fragment compatibility, obtained from a Bayesian classification procedure. A simplex optimization was used to optimize the sequence-based terms for the goal of alignment and model quality and to balance the sequence and structural contributions against each other. Both sequence and structural terms operate with sequence profiles.

Amino Acid Substitution↗
PubMed2004

Bellerophon: a program to detect chimeric sequences in multiple sequence alignments.

SUMMARY: Bellerophon is a program for detecting chimeric sequences in multiple sequence datasets by an adaption of partial treeing analysis. Bellerophon was specifically developed to detect 16S rRNA gene chimeras in PCR-clone libraries of environmental samples but can be applied to other nucleotide sequence alignments. AVAILABILITY: Bellerophon is available as an interactive web server at http://foo.maths.uq.edu.au/~huber/bellerophon.pl

Algorithms↗
PubMed2004

Fast structure-based assignment of 15N HSQC spectra of selectively 15N-labeled paramagnetic proteins.

A novel strategy for fast NMR resonance assignment of (15)N HSQC spectra of proteins is presented. It requires the structure coordinates of the protein, a paramagnetic center, and one or more residue-selectively (15)N-labeled samples. Comparison of sensitive undecoupled (15)N HSQC spectra recorded of paramagnetic and diamagnetic samples yields data for every cross-peak on pseudocontact shift, paramagnetic relaxation enhancement, cross-correlation between Curie-spin and dipole-dipole relaxation, and residual dipolar coupling. Comparison of these four different paramagnetic quantities with predictions from the three-dimensional structure simultaneously yields the resonance assignment and the anisotropy of the susceptibility tensor of the paramagnetic center. The method is demonstrated with the 30 kDa complex between the N-terminal domain of the epsilon subunit and the theta subunit of Escherichia coli DNA polymerase III. The program PLATYPUS was developed to perform the assignment, provide a measure of reliability of the assignment, and determine the susceptibility tensor anisotropy.

Algorithms↗