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Biomedical subjects

G Vriend

Publications and source records attributed to G Vriend.

At least 19 recordsLinked to original sources
PubMed2005

MRS: a fast and compact retrieval system for biological data.

The biological data explosion of the 'omics' era requires fast access to many data types in rapidly growing data banks. The MRS server allows for very rapid queries in a large number of flat-file data banks, such as EMBL, UniProt, OMIM, dbEST, PDB, KEGG, etc. This server combines a fast and reliable backend with a very user-friendly implementation of all the commonly used information retrieval facilities. The MRS server is freely accessible at http://mrs.cmbi.ru.nl/. Moreover, the MRS software is freely available at http://mrs.cmbi.ru.nl/download/ for those interested in making their own data banks available via a web-based server.

Databases, Genetic↗
PubMed2004

Heavier-than-air flying machines are impossible.

Many G protein-coupled receptor (GPCR) models have been built over the years. The release of the structure of bovine rhodopsin in August 2000 enabled us to analyze models built before that period to learn more about the models we build today. We conclude that the GPCR modelling field is riddled with 'common knowledge' similar to Lord Kelvin's remark in 1895 that "heavier-than-air flying machines are impossible", and we summarize what we think are the (im)possibilities of modelling GPCRs using the coordinates of bovine rhodopsin as a template. Associated WWW pages: www.gpcr.org/articles/2003_mod

Amino Acid Sequence↗
PubMed2002

Correlated mutation analyses on very large sequence families.

The 'omics era' (the era of genomics, proteomics, and so forth) is marked by a flood of data that need to be interpreted to become useful information. Thanks to genome sequencing projects, large numbers of sequence families with more than a thousand members each are now available. Novel analytical techniques are needed to deal with this avalanche of sequence data. Sequence entropy is a measure of the information present in an alignment, whereas sequence variability represents the mutational flexibility at a particular position. Entropy versus variability plots can reveal the roles of groups of residues in the overall function of a protein. Such roles can be as part of the main active site, part of a modulator binding site, or transduction of a signal between those sites. Residues that are involved in a common function tend to stay conserved as a group, but when they mutate, they tend to mutate together. Correlated mutation analysis can detect groups of residue positions that show this behaviour. The combination of entropy, variability and correlation is a powerful tool to convert sequence data into useful information. This analysis can, for example, detect the key residues involved in cooperativity in globins, the switch regions in ras-like proteins and the calcium binding and signalling residues in serine proteases. We have extrapolated from these three classes of structurally and functionally well-described proteins to G-protein-coupled receptors (GPCRs). We can detect the residues in the main functional site in GPCRs that are responsible for G-protein coupling, the residues in the endogenous agonist binding site, and the residues in between that transduce the signal to and fro between these sites. The results are discussed in the light of a simple two-step evolutionary model for the development of functional proteins.

Amino Acid Sequence↗
PubMed2002

Completion and refinement of 3-D homology models with restricted molecular dynamics: application to targets 47, 58, and 111 in the CASP modeling competition and posterior analysis.

A method is presented to refine models built by homology by the use of restricted molecular dynamics (MD) techniques. The basic idea behind this method is the use of structure validation software to determine for each residue the likelihood that it is modeled correctly. This information is used to determine constraints and restraints in an MD simulation including explicit solvent molecules, which is used for model refinement. The procedure is based on the idea that residues that the validation software identifies as correctly positioned should be strongly constrained or restrained in the MD simulations, whereas residues that are likely to be positioned wrongly should move freely. Two different protocols are compared: one (applied to CASP3 target T58) using full structural constraints with separate optimization of each short fragment and the other (applied to T47) allowing some freedom using harmonic restraining potentials, with automatic optimization of the whole molecule. Structures along the MD trajectory that scored best in structural checks were selected for the construction of models that appeared to be successful in the CASP3 competition. Model refinement with MD in general leads to a model that is less like the experimental structure (Levitt et al. Nature Struct Biol 1999;6:108-111). Actually, refined T47 was slightly improved compared to the starting model; changes in model T58 led not to further enhancement. After the X-ray structure of the modeled proteins became known, the procedure was evaluated for two targets (T47 and the CASP4 target T111) by comparing a long simulation in water with the experimental target structures. It was found that structural improvements could be obtained on a nanosecond time scale by allowing appropriate freedom in the simulation. Structural checks applied to fast fluctuations do not appear to be informative for the correctness of the structure. However, both a simple hydrogen bond count and a simple compactness measure, if averaged over times of typically 300 ps, correlate well with structural correctness and we suggest that criteria based on these properties may be used in computational folding strategies.

Amino Acid Sequence↗
PubMed2001

Amyloid-like fibrils from an 18-residue peptide analogue of a part of the central domain of the B-family of silkmoth chorion proteins.

Chorion is the major component of silkmoth eggshell. More than 95% of its dry mass consists of the A and B families of low molecular weight structural proteins, which have remarkable mechanical and chemical properties, and protect the oocyte and the developing embryo from the environment. We present data from negative staining, Congo red binding, X-ray diffraction, Fourier transform-Raman, attenuated total reflectance infrared spectroscopy and modelling studies of a synthetic peptide analogue of a part of the central domain of the B family of silkmoth chorion proteins, indicating that this peptide folds and self-assembles, forming amyloid-like fibrils. These results support further our proposal, based on experimental data from a synthetic peptide analogue of the central domain of the A family of chorion proteins, that silkmoth chorion is a natural, protective amyloid [Iconomidou et al., FEBS Lett. 479 (2000) 141-145].

Amyloid↗
PubMed2001

Optimizing the hydrogen-bond network in Poisson-Boltzmann equation-based pK(a) calculations.

pK(a) calculation methods that are based on finite difference solutions to the Poisson-Boltzmann equation (FDPB) require that energy calculations be performed for a large number of different protonation states of the protein. Normally, the differences between these protonation states are modeled by changing the charges on a few atoms, sometimes the differences are modeled by adding or removing hydrogens, and in a few cases the positions of these hydrogens are optimized locally. We present an FDPB-based pK(a) calculation method in which the hydrogen-bond network is globally optimized for every single protonation state used. This global optimization gives a significant improvement in the accuracy of calculated pK(a) values, especially for buried residues. It is also shown that large errors in calculated pK(a) values are often due to structural artifacts induced by crystal packing. Optimization of the force fields and parameters used in pK(a) calculations should therefore be performed with X-ray structures that are corrected for crystal artifacts.

Amino Acid Sequence↗
PubMed2001

Conserved role of a complement-like protein in phagocytosis revealed by dsRNA knockout in cultured cells of the mosquito, Anopheles gambiae.

We characterize a novel hemocyte-specific acute phase glycoprotein from the malaria vector, Anopheles gambiae. It shows substantial structural and functional similarities, including the highly conserved thioester motif, to both a central component of mammalian complement system, factor C3, and to a pan-protease inhibitor, alpha2-macroglobulin. Most importantly, this protein serves as a complement-like opsonin and promotes phagocytosis of some Gram-negative bacteria in a mosquito hemocyte-like cell line. Chemical inactivation by methylamine and depletion by double-stranded RNA knockout demonstrate that this function is dependent on the internal thioester bond. This evidence of a complement-like function in a protostome animal adds substantially to the accumulating evidence of a common ancestry of immune defenses in insects and vertebrates.

Animals↗
PubMed2001

Collecting and harvesting biological data: the GPCRDB and NucleaRDB information systems.

The amount of genomic and proteomic data that is entered each day into databases and the experimental literature is outstripping the ability of experimental scientists to keep pace. While generic databases derived from automated curation efforts are useful, most biological scientists tend to focus on a class or family of molecules and their biological impact. Consequently, there is a need for molecular class-specific or other specialized databases. Such databases collect and organize data around a single topic or class of molecules. If curated well, such systems are extremely useful as they allow experimental scientists to obtain a large portion of the available data most relevant to their needs from a single source. We are involved in the development of two such databases with substantial pharmacological relevance. These are the GPCRDB and NucleaRDB information systems, which collect and disseminate data related to G protein-coupled receptors and intra-nuclear hormone receptors, respectively. The GPCRDB was a pilot project aimed at building a generic molecular class-specific database capable of dealing with highly heterogeneous data. A first version of the GPCRDB project has been completed and it is routinely used by thousands of scientists. The NucleaRDB was started recently as an application of the concept for the generalization of this technology. The GPCRDB is available via the WWW at http://www.gpcr.org/7tm/ and the NucleaRDB at http://www.receptors.org/NR/.

Binding, Competitive↗
PubMed2001

A sequence and structural study of transmembrane helices.

A comparison is made between the distribution of residue preferences, three dimensional nearest neighbour contacts, preferred rotamers, helix-helix crossover angles and peptide bond angles in three sets of proteins: a non-redundant set of accurately determined globular protein structures, a set of four-helix bundle structures and a set of membrane protein structures. Residue preferences for the latter two sets may reflect overall helix stabilising propensities but may also highlight differences arising out of the contrasting nature of the solvent environments in these two cases. The results bear out the expectation that there may be differences between residue type preferences in membrane proteins and in water soluble globular proteins. For example, the beta-branched residue types valine and isoleucine are considerably more frequently encountered in membrane helices. Likewise, glycine and proline. residue types normally associated with 'helix-breaking' propensity are found to be relatively more common in membrane helices. Three dimensional nearest neighbour contacts along the helix, preferred rotamers, and peptide bond angles are very similar in the three sets of proteins as far as can be ascertained within the limits of the relatively low resolution of the membrane proteins dataset. Crossing angles for helices in the membrane protein set resemble the four helix bundle set more than the general non-redundant set, but in contrast to both sets they have smaller crossing angles consistent with the dual requirements for the helices to form a compact structure while having to span the membrane. In addition to the pairwise packing of helices we investigate their global packing and consider the question of helix supercoiling in helix bundle proteins.

Amino Acid Sequence↗
PubMed2001

The effect of changing the hydrophobic S1' subsite of thermolysin-like proteases on substrate specificity.

The hydrophobic S1' subsite is one of the major determinants of the substrate specificity of thermolysin and related M4 family proteases. In the thermolysin-like protease (TLP) produced by Bacillus stearothermophilus (TLP-ste), the hydrophobic S1' subsite is mainly formed by Phe130, Phe133, Val139 and Leu202. In the present study, we have examined the effects of replacing Leu202 by smaller (Gly, Ala, Val) and larger (Phe, Tyr) hydrophobic residues. The mutational effects showed that the wild-type S1' pocket is optimal for binding leucine side chains. Reduction of the size of residue 202 resulted in a higher efficiency towards substrates with Phe in the P1' position. Rather unexpectedly, the Leu202-->Phe and Leu202-->Tyr mutations, which were expected to decrease the size of the S1' subsite, resulted in a large increase in activity towards dipeptide substrates with Phe in the P1' position. This is probably due to the fact that 202Phe and 202Tyr adopt a second possible rotamer that opens up the subsite compared to Leu202, and also favours interactions with the substrate. To validate these results, we constructed variants of thermolysin with changes in the S1' subsite. Thermolysin and TLP-ste variants with identical S1' subsites were highly similar in terms of their preference for Phe vs. Leu in the P1' position.

Amino Acid Substitution↗
PubMed2001

The determinants of alpha-amylase pH-activity profiles.

The glycosyl hydrolases present a large family of enzymes that are of great significance for industry. Consequently, there is considerable interest in engineering the enzymes in this family for optimal performance under a range of very diverse conditions. Until recently, tailoring glycosyl hydrolases for specific industrial processes mainly involved stability engineering, but lately there has also been considerable interest in engineering their pH-activity profiles. We mutated four neutral residues (N190, F290, N326 and Q360) in the chimeric Bacillus Ba2 alpha-amylase to both charged and neutral amino acids. The results show that the pH-activity profile of the Ba2 alpha-amylase can be changed by inserting charged residues close to the active site. The changes in the pH-activity profile for these neutral --> charged mutations do not, however, correlate with the predictions from calculations of the p K(a) values of the active site residues. More surprisingly, the neutral --> neutral mutations change the pH-activity profile as much as the neutral --> charged mutations. From these results, it is concluded that factors other than electrostatics, presumably the dynamic aspects of the active site, are important for the shape of the pH-activity profiles of the alpha-amylases.

Amino Acids↗
PubMed2000

Synergism with the coactivator OBF-1 (OCA-B, BOB-1) is mediated by a specific POU dimer configuration.

POU domain proteins contain a bipartite DNA binding domain divided by a flexible linker that enables them to adopt various monomer configurations on DNA. The versatility of POU protein operation is additionally conferred at the dimerization level. The POU dimer formed on the PORE (ATTTGAAATGCAAAT) can recruit the transcriptional coactivator OBF-1, whereas POU dimers formed on the consensus MORE (ATGCATATGCAT) or on MOREs from immunoglobulin heavy chain promoters (AT[G/A][C/A]ATATGCAA) fail to interact. An interaction with OBF-1 is precluded since the same Oct-1 residues that form the MORE dimerization interface are also used for OBF-1/Oct-1 interactions on the PORE. Our findings provide a paradigm of how specific POU dimer assemblies can differentially recruit a coregulatory activity with distinct transcriptional readouts.

Amino Acid Motifs↗
PubMed2000

Receptors coupling to G proteins: is there a signal behind the sequence?

Upon the binding of their ligands, G protein-coupled receptors couple to the heterotrimeric G proteins to transduce a signal. One receptor family may couple to a single G protein subtype and another family to several ones. Is there a signal in the receptor sequence that can give an indication of the G protein subtype selectivity? We used a sequence analysis method on biogenic amine and adenosine receptors and concluded that a weak signal can be detected in receptor families where specialization for coupling to a given G protein occurred during a recent divergent evolutionary process. Proteins 2000;41:448-459.

Algorithms↗
PubMed2000

Amyloids protect the silkmoth oocyte and embryo.

Chorion is the major component of silkmoth eggshell. More than 95% of its dry mass consists of proteins that have remarkable mechanical and chemical properties protecting the oocyte and the developing embryo from a wide range of environmental hazards. We present data from electron microscopy (negative staining and shadowing), X-ray diffraction and modeling studies of synthetic peptide analogues of silkmoth chorion proteins indicating that chorion is a natural amyloid. The folding and self-assembly models of chorion peptides strongly support the beta-sheet helix model of amyloid fibrils proposed recently by Blake and Serpell [Structure 4 (1996) 989-998].

Amino Acid Sequence↗
PubMed1999

Validation of nuclear magnetic resonance structures of proteins and nucleic acids: hydrogen geometry and nomenclature.

A statistical analysis is reported of 1,200 of the 1,404 nuclear magnetic resonance (NMR)-derived protein and nucleic acid structures deposited in the Protein Data Bank (PDB) before 1999. Excluded from this analysis were the entries not yet fully validated by the PDB and the more than 100 entries that contained < 95% of the expected hydrogens. The aim was to assess the geometry of the hydrogens in the remaining structures and to provide a check on their nomenclature. Deviations in bond lengths, bond angles, improper dihedral angles, and planarity with respect to estimated values were checked. More than 100 entries showed anomalous protonation states for some of their amino acids. Approximately 250,000 (1.7%) atom names differed from the consensus PDB nomenclature. Most of the inconsistencies are due to swapped prochiral labeling. Large deviations from the expected geometry exist for a considerable number of entries, many of which are average structures. The most common causes for these deviations seem to be poor minimization of average structures and an improper balance between force-field constraints for experimental and holonomic data. Some specific geometric outliers are related to the refinement programs used. A number of recommendations for biomolecular databases, modeling programs, and authors submitting biomolecular structures are given.

Data Interpretation, Statistical↗
PubMed1999

Heterozygous germline mutations in the p53 homolog p63 are the cause of EEC syndrome.

EEC syndrome is an autosomal dominant disorder characterized by ectrodactyly, ectodermal dysplasia, and facial clefts. We have mapped the genetic defect in several EEC syndrome families to a region of chromosome 3q27 previously implicated in the EEC-like disorder, limb mammary syndrome (LMS). Analysis of the p63 gene, a homolog of p53 located in the critical LMS/EEC interval, revealed heterozygous mutations in nine unrelated EEC families. Eight mutations result in amino acid substitutions that are predicted to abolish the DNA binding capacity of p63. The ninth is a frameshift mutation that affects the p63alpha, but not p63beta and p63gamma isotypes. Transactivation studies with these mutant p63 isotypes provide a molecular explanation for the dominant character of p63 mutations in EEC syndrome.

Abnormalities, Multiple↗
PubMed1999

Probing the unfolding region in a thermolysin-like protease by site-specific immobilization.

Protein stabilization by immobilization has been proposed to be most effective if the protein is attached to the carrier at that region where unfolding is initiated. To probe this hypothesis, we have studied the effects of site-specific immobilization on the thermal stability of mutants of the thermolysin-like protease from Bacillus stearothermophilus (TLP-ste). This enzyme was chosen because previous studies had revealed which parts of the molecule are likely to be involved in the early steps of thermal unfolding. Cysteine residues were introduced by site-directed mutagenesis into various positions of a cysteine-free variant of TLP-ste. The mutant enzymes were immobilized in a site-specific manner onto Activated Thiol-Sepharose. Two mutants (T56C, S65C) having their cysteine in the proposed unfolding region of TLP-ste showed a 9- and 12-fold increase in half-lives at 75 degrees C due to immobilization. The stabilization by immobilization was even larger (33-fold) for the T56C/S65C double mutant enzyme. In contrast, mutants containing cysteines in other parts of the TLP-ste molecule (N181C, S218C, T299C) showed only small increases in half-lives due to immobilization (maximum 2.5-fold). Thus, the stabilization obtained by immobilization was strongly dependent on the site of attachment. It was largest when TLP-ste was fixed to the carrier through its postulated unfolding region. The concept of the unfolding region may be of general use for the design of strategies to stabilize proteins.

Cysteine↗