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G Vriend

Publications and source records attributed to G Vriend.

At least 37 records · Page 2Linked to original sources
PubMed1999

Conformational transitions accompanying oligomerization of yeast alcohol oxidase, a peroxisomal flavoenzyme.

Alcohol oxidase (AO) is a homo-octameric flavoenzyme which catalyzes methanol oxidation in methylotrophic yeasts. AO protein is synthesized in the cytosol and subsequently sorted to peroxisomes where the active enzyme is formed. To gain further insight in the molecular mechanisms involved in AO activation, we studied spectroscopically native AO from Hansenula polymorpha and Pichia pastoris and three putative assembly intermediates. Fluorescence studies revealed that both Trp and FAD are suitable intramolecular markers of the conformation and oligomeric state of AO. A direct relationship between dissociation of AO octamers and increase in Trp fluorescence quantum yield and average fluorescence lifetime was found. The time-resolved fluorescence of the FAD cofactor showed a rapid decay component which reflects dynamic quenching due to the presence of aromatic amino acids in the FAD-binding pocket. The analysis of FAD fluorescence lifetime profiles showed a remarkable resemblance of pattern for purified AO and AO present in intact yeast cells. Native AO contains a high content of ordered secondary structure which was reduced upon FAD-removal. Dissociation of octamers into monomers resulted in a conversion of beta-sheets into alpha-helices. Our results are explained in relation to a 3D model of AO, which was built based on the crystallographic data of the homologous enzyme glucose oxidase from Aspergillus niger. The implications of our results for the current model of the in vivo AO assembly pathway are discussed.

Alcohol Oxidoreductases↗
PubMed1999

Conformational changes in the chaperonin GroEL: new insights into the allosteric mechanism.

Conformational changes are known to play a crucial role in the function of the bacterial GroE chaperonin system. Here, results are presented from an essential dynamics analysis of known experimental structures and from computer simulations of GroEL using the CONCOORD method. The results indicate a possible direct form of inter-ring communication associated with internal fluctuations in the nucleotide-binding domains upon nucleotide and GroES binding that are involved in the allosteric mechanism of GroEL. At the level of conformational transitions in entire GroEL rings, nucleotide-induced structural changes were found to be distinct and in principle uncoupled from changes occurring upon GroES binding. However, a coupling is found between nucleotide-induced conformational changes and GroES-mediated transitions, but only in simulations of GroEL double rings, and not in simulations of single rings. This provides another explanation for the fact that GroEL functions a double ring system.

Allosteric Regulation↗
PubMed1999

A low resolution model for the interaction of G proteins with G protein-coupled receptors.

A model is presented for the interaction between G proteins and G protein-coupled receptors. The model is based on the fact that this interaction shows little specificity and thus conserved parts of the G proteins have to interact with conserved parts of the receptors. These parts are a conserved negative residue in the G protein, a fully conserved arginine in the receptor and a series of residues that are not conserved but always hydrophobic like the hydrophobic side of the C-terminal helix of the G protein and the hydrophobic side of a helix in the C-terminal domain of the receptor. Other, mainly cytosolic, factors determine the specificity and regulation of this interaction. The relation between binding and activation will be shown. A large body of experimental evidence supports this model. Despite the fact that the model does not provide atomic resolution, it can be used to explain some experimental data that would otherwise seem inexplicable, and it suggests experiments for its falsification or verification.

Amino Acid Sequence↗
PubMed1999

Structural and mutagenesis studies of leishmania triosephosphate isomerase: a point mutation can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power.

The dimeric enzyme triosephosphate isomerase (TIM) has a very tight and rigid dimer interface. At this interface a critical hydrogen bond is formed between the main chain oxygen atom of the catalytic residue Lys13 and the completely buried side chain of Gln65 (of the same subunit). The sequence of Leishmania mexicana TIM, closely related to Trypanosoma brucei TIM (68% sequence identity), shows that this highly conserved glutamine has been replaced by a glutamate. Therefore, the 1.8 A crystal structure of leishmania TIM (at pH 5.9) was determined. The comparison with the structure of trypanosomal TIM shows no rearrangements in the vicinity of Glu65, suggesting that its side chain is protonated and is hydrogen bonded to the main chain oxygen of Lys13. Ionization of this glutamic acid side chain causes a pH-dependent decrease in the thermal stability of leishmania TIM. The presence of this glutamate, also in its protonated state, disrupts to some extent the conserved hydrogen bond network, as seen in all other TIMs. Restoration of the hydrogen bonding network by its mutation to glutamine in the E65Q variant of leishmania TIM results in much higher stability; for example, at pH 7, the apparent melting temperature increases by 26 degrees C (57 degrees C for leishmania TIM to 83 degrees C for the E65Q variant). This mutation does not affect the kinetic properties, showing that even point mutations can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power at the mesophilic temperature.

Animals↗
PubMed1999

Improving macromolecular electrostatics calculations.

Electrostatic interactions play a key role in many aspects of protein engineering. Consequently, much effort has been put into the design of software for calculating electrostatic fields around macromolecules. We show that optimization of hydrogen bonding networks can improve both the results of pK(a) calculations and the results of electrostatic calculations performed by commonly used programs such as DelPhi. Further optimization can often be achieved by flipping the side chains of asparagine, histidine and glutamine around their chi2, chi2 and chi3 torsion angles, respectively, when this improves the local hydrogen bonding network. These optimizations are applied to some well characterized proteins: BPTI, hen egg white lysozyme and superoxide dismutase. A search for flipped residues in the PDB revealed that significant improvements in electrostatic calculations in or near the active site of enzymes can be expected for about one quarter of all enzymes in the PDB.

Animals↗
PubMed1998

Early steps in the unfolding of thermolysin-like proteases.

Several series of site-directed mutations in thermolysin-like proteases are presented that show remarkable nonadditivity in their effect on thermal stability. A simple model is proposed that relates this nonadditivity to the occurrence of independent partial unfolding processes that occur in parallel at elevated temperatures. To prove this model, a thermolysin-like protease was designed in which two mutations located approximately 35 A apart in the structure individually exert small stabilizing effects of 2.3 and 4. 1 degreesC, respectively, but when combined stabilize the protease by 14.6 degreesC. This overadditivity, which follows directly from the model, confirms that unfolding of this engineered protease starts in parallel at two different regions of the protein.

Bacillus↗
PubMed1998

New POU dimer configuration mediates antagonistic control of an osteopontin preimplantation enhancer by Oct-4 and Sox-2.

The POU transcription factor Oct-4 is expressed specifically in the germ line, pluripotent cells of the pregastrulation embryo and stem cell lines derived from the early embryo. Osteopontin (OPN) is a protein secreted by cells of the preimplantation embryo and contains a GRGDS motif that can bind to specific integrin subtypes and modulate cell adhesion/migration. We show that Oct-4 and OPN are coexpressed in the preimplantation mouse embryo and during differentiation of embryonal cell lines. Immunoprecipitation of the first intron of OPN (i-opn) from covalently fixed chromatin of embryonal stem cells by Oct-4-specific antibodies indicates that Oct-4 binds to this fragment in vivo. The i-opn fragment functions as an enhancer in cell lines that resemble cells of the preimplantation embryo. Furthermore, it contains a novel palindromic Oct factor recognition element (PORE) that is composed of an inverted pair of homeodomain-binding sites separated by exactly 5 bp (ATTTG +5 CAAAT). POU proteins can homo- and heterodimerize on the PORE in a configuration that has not been described previously. Strong transcriptional activation of the OPN element requires an intact PORE. In contrast, the canonical octamer overlapping with the downstream half of the PORE is not essential. Sox-2 is a transcription factor that contains an HMG box and is coexpressed with Oct-4 in the early mouse embryo. Sox-2 represses Oct-4 mediated activation of i-opn by way of a canonical Sox element that is located close to the PORE. Repression depends on a carboxy-terminal region of Sox-2 that is outside of the HMG box. Expression, DNA binding, and transactivation data are consistent with the hypothesis that OPN expression is regulated by Oct-4 and Sox-2 in preimplantation development.

Amino Acid Sequence↗
PubMed1998

Probing catalytic hinge bending motions in thermolysin-like proteases by glycine --> alanine mutations.

The active site of thermolysin-like proteases (TLPs) is located at the bottom of a cleft between the N- and C-terminal domains. Crystallographic studies have shown that the active-site cleft is more closed in ligand-binding TLPs than in ligand-free TLPs. Accordingly, it has been proposed that TLPs undergo a hinge-bending motion during catalysis resulting in "closure" and "opening" of the active-site cleft. Two hinge regions have been proposed. One is located around a conserved glycine 78; the second involves residues 135 and 136. The importance of conserved glycine residues in these hinge regions was studied experimentally by analyzing the effects of Gly --> Ala mutations on catalytic activity. Eight such mutations were made in the TLP of Bacillus stearothermophilus (TLP-ste) and their effects on activity toward casein and various peptide substrates were determined. Only the Gly78Ala, Gly136Ala, and Gly135Ala + Gly136Ala mutants decreased catalytic activity significantly. These mutants displayed a reduction in kcat/Km for 3-(2-furylacryloyl)-L-glycyl-L-leucine amide of 73%, 62%, and 96%, respectively. Comparisons of effects on kcat/Km for various substrates with effects on the Ki for phosphoramidon suggested that the mutation at position 78 primarily had an effect on substrate binding, whereas the mutations at positions 135 and 136 primarily influence kcat. The apparent importance of conserved glycine residues in proposed hinge-bending regions for TLP activity supports the idea that hinge-bending is an essential part of catalysis.

Alanine↗
PubMed1998

A single calcium binding site is crucial for the calcium-dependent thermal stability of thermolysin-like proteases.

Thermostable thermolysin-like proteases (TLPs), such as the TLP of Bacillus stearothermophilus CU-21 (TLP-ste), bind calcium in one double (Ca1,2) and two single (Ca3, Ca4) calcium binding sites. The single sites are absent in thermolabile TLPs, suggesting that they are determinants of (variation in) TLP stability. Mutations in the Ca3 and Ca4 sites of TLP-ste indeed reduced thermal stability, but only mutations in the Ca3 site affected the calcium-dependence of stability. The predominant effect of the Ca3 site results from the fact that the Ca3 site is part of a region of TLP-ste, which unfolding is crucial for thermal inactivation. Thermal inactivation is not caused by the absence of calcium from the Ca3 site per se, but rather by unfolding of a region of TLP-ste for which stability depends on the occupancy of the Ca3 site. In accordance with this concept is the observation that the effects of mutations in the Ca3 site could be compensated by stabilizing mutations near this site. In addition, it was observed that the contribution of calcium binding to the Ca3 was substantially reduced in extremely stable TLP-ste variants containing multiple stabilizing mutations in the Ca3 region. Apparently, in these latter variants, unfolding of the Ca3 region contributes little to the overall process of thermal inactivation.

Amino Acid Sequence↗
PubMed1998

Engineering an enzyme to resist boiling.

In recent years, many efforts have been made to isolate enzymes from extremophilic organisms in the hope to unravel the structural basis for hyperstability and to obtain hyperstable biocatalysts. Here we show how a moderately stable enzyme (a thermolysin-like protease from Bacillus stearothermophilus, TLP-ste) can be made hyperstable by a limited number of mutations. The mutational strategy included replacing residues in TLP-ste by residues found at equivalent positions in naturally occurring, more thermostable variants, as well as rationally designed mutations. Thus, an extremely stable 8-fold mutant enzyme was obtained that was able to function at 100 degrees C and in the presence of denaturing agents. This 8-fold mutant contained a relatively large number of mutations whose stabilizing effect is generally considered to result from a reduction of the entropy of the unfolded state ("rigidifying" mutations such as Gly --> Ala, Ala --> Pro, and the introduction of a disulfide bridge). Remarkably, whereas hyperstable enzymes isolated from natural sources often have reduced activity at low temperatures, the 8-fold mutant displayed wild-type-like activity at 37 degrees C.

Amino Acid Substitution↗
PubMed1998

GPCRDB: an information system for G protein-coupled receptors.

The GPCRDB is a G protein-coupled receptor (GPCR) database system aimed at the collection and dissemination of GPCR related data. It holds sequences, mutant data and ligand binding constants as primary (experimental) data. Computationally derived data such as multiple sequence alignments, three dimensional models, phylogenetic trees and two dimensional visualization tools are added to enhance the database's usefulness. The GPCRDB is an EU sponsored project aimed at building a generic molecular class specific database capable of dealing with highly heterogeneous data. GPCRs were chosen as test molecules because of their enormous importance for medical sciences and due to the availability of so much highly heterogeneous data. The GPCRDB is available via the WWW at http://www.gpcr.org/7tm

Computer Communication Networks↗
PubMed1998

The mutations in FGFR2-associated craniosynostoses are clustered in five structural elements of immunoglobulin-like domain III of the receptor.

Exons 5 and 7 of the fibroblast growth factor receptor 2 (FGFR2) gene code for immunoglobulin-like domain III (IgIII) and for the region connecting the second and the third Ig domain of the receptor. Numerous mutations in these two exons have been shown to cause various craniosynostotic syndromes. Here, we describe three previously unrecognized mutations at amino acid positions 276, 301, and 314, in one nonspecific craniosynostosis and in two Crouzon patients. We also present a polypeptide model of IgIII of FGFR2. The known mutations involve five distinct structural elements of the receptor. The changes within these elements affect receptor function by various mechanisms, including altered dimerization, truncation, increased mobility between Ig domains, disintegration of IgIII, and alteration of the ligand-binding site.

Craniofacial Dysostosis↗
PubMed1998

Homology modeling, model and software evaluation: three related resources.

MOTIVATION: Homology modeling is rapidly becoming the method of choice for obtaining three-dimensional coordinates for proteins because genome projects produce sequences at a much higher rate than NMR and X-ray laboratories can solve the three-dimensional structures. The quality of protein models will not be immediately clear to novices and support with the evaluation seems to be needed. Expert users are sometimes interested in evaluating the quality of modeling programs rather than the quality of the models themselves. RESULTS: Three servers have been made available to the scientific community: a homology modeling server, a model quality evaluation server and a server that evaluates models built of proteins for which the structure is already known, thereby implicitly evaluating the quality of the modeling program. AVAILABILITY: The modeling-related servers and several structure analysis servers are freely available at http://swift.embl-heidelberg.de/servers/ CONTACT: gert.vriend@embl-heidelberg.de

Computational Biology↗
PubMed1998

Rendering one autolysis site in Bacillus subtilis neutral protease resistant to cleavage reveals a new fission.

Autolytic degradation of the thermolysin-like proteinase of Bacillus subtilis (TLP-sub) is responsible for the irreversible inactivation of the enzyme at elevated temperatures. Previously we have reported five cleavage sites in TLP-sub [Van den Burg et al. (1990) Biochem. J. 272, 93-97]. In an attempt to render the enzyme less susceptible to autolytic breakdown, one of the fission sites, located between Leu-156 and Ile-157, was modified by replacing Ile-157, C-terminally located with respect to the fission site, by an Asp residue. Aspartic acid is less preferred at this position with respect to the substrate preference of TLP-sub. Modelling studies indicated that this mutation was unlikely to cause conformational changes in the enzyme. Although the 156-157 fission was not observed in the mutant enzyme, a new fission site, between Gly-148 and Val-149, was now observed.

Amino Acid Sequence↗
PubMed1998

The interaction of class B G protein-coupled receptors with their hormones.

In common with many G protein-coupled receptors, dysfunction in members of the Class B or glucagon-like receptors can elicit a wide spectrum of disease related activities. Consequently, they are potential targets in many different areas of pharmacological research. Unlike the class A or rhodopsin-like receptors, for which at least some structural similarity to bacteriorhodopsin has been detected, absolutely no structural information is available for the Class B G protein-coupled receptors. We present a computational study that exploits the experimental work performed by evolution to indicate residues that are potentially involved in ligand binding in the Class B G protein-coupled receptors. We perform an analysis of mutations that occurred in a correlated fashion between the receptors and their peptidic ligands. The inference that the residues detected in this manner are involved in a direct interaction between the receptor and the ligand is in good agreement with the mutation studies that have already been published.

Amino Acid Sequence↗
PubMed1997

Mutational analysis of a surface area that is critical for the thermal stability of thermolysin-like proteases.

Site-directed mutagenesis was used to assess the contribution of individual residues and a bound calcium in the 55-69 region of the thermolysin-like protease of Bacillus stearothermophilus (TLP-ste) to thermal stability. The importance of the 55-69 region was reflected by finding that almost all mutations had drastic effects on stability. These effects (both stabilizing and destabilizing) were obtained by mutations affecting main chain flexibility, as well as by mutations affecting the interaction between the 55-69 region and the rest of the protease molecule. The calcium-dependency of stability could be largely abolished by mutating one of its ligands (Asp57 or Asp59). In the case of the Asp57-->Ser mutation, the accompanying loss in stability was modest compared with the effects of other destabilizing mutations or the effects of (combinations of) stabilizing mutations. The detailed knowledge of the stability-determining region of TLP-ste permits effective rational design of stabilizing mutations, which, presumably, are also useful for related TLP such as thermolysin. This is demonstrated by the successful design of a stabilizing salt bridge involving residues 65 and 11.

Amino Acid Sequence↗
PubMed1997

Analysis of the black-eyed pea trypsin and chymotrypsin inhibitor-alpha-chymotrypsin complex.

The black-eyed pea trypsin and chymotrypsin inhibitor (BTCI) is a member of the Bowman-Birk protease inhibitor (BBI) family. The three-dimensional model of the BTCI-chymotrypsin complex was built based on the homology to Bowman-Birk inhibitors with known structures. An extensive theoretical and experimental study of these known structures has been performed. The model confirms the ideas about Bowman-Birk inhibitor structure-function relations and agrees well with our experimental data (circular dichroism, IR and light scattering). The electrostatic potentials at the enzyme-inhibitor contact surface reveal a pattern of complementary electrostatic potentials from which mutations can be inferred that could give these inhibitors an altered specificity.

Amino Acid Sequence↗