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G Vriend

Publications and source records attributed to G Vriend.

At least 73 records · Page 4Linked to original sources
PubMed1995

Structural determinants of the stability of thermolysin-like proteinases.

Thermolysin is a member of a family of homologous proteinases which differ in their resistance to thermally induced unfolding and subsequent autolytic degradation. Site-directed mutagenesis studies of the thermolysin-like proteinase (TLP) from Bacillus stearothermophilus (TLP-ste) show that its reduced resistance to thermally induced autolysis, as compared to thermolysin, is due to only some of the 44 naturally occurring amino-acid differences between them. In fact TLP-ste becomes more resistant than thermolysin by mutation of just a few of these amino-acids. The crucial differences are all localized to a solvent-exposed region in the N-terminal domain of TLP-ste.

Amino Acid Sequence↗
PubMed1995

Progesterone binding to uteroglobin: two alternative orientations of the ligand.

Progesterone binding to a homodimer of uteroglobin takes place in a hydrophobic cavity formed by the two subunits. Previous mutational analyses have shown that the tyrosine (21 and 21') and threonine (60 and 60') residues of the uteroglobin dimer are directly involved in progesterone binding. To analyze the contribution of each of the two tyrosines and threonines in the dimer, we have constructed a covalently linked uteroglobin dimer (UGcl) by fusing two uteroglobin cDNAs via a synthetic linker sequence. Escherichia coli expressed UGcl bound progesterone with the same affinity as the native dimeric protein. Replacement of both tyrosines by phenylalanines abolished progesterone binding. Replacement of either the C-terminal tyrosine 21 or the N-terminal tyrosine 21' separately, reduced the affinity for progesterone 3- to 4-fold, suggesting that both tyrosines participate in progesterone binding. In contrast, substitutions of the threonine residues of the C- or N-terminal moities had no effect, whereas the replacement of both threonines reduced the affinity for progesterone 2- to 3-fold. These data, together with computer models, suggest that progesterone docks in the internal binding pocket of uteroglobin in two different orientations.

Amino Acid Sequence↗
PubMed1995

Potential ligand-binding residues in rat olfactory receptors identified by correlated mutation analysis.

A family of G-protein-coupled receptors is believed to mediate the recognition of odor molecules. In order to identify potential ligand-binding residues, we have applied correlated mutation analysis to receptor sequences from the rat. This method identifies pairs of sequence positions where residues remain conserved or mutate in tandem, thereby suggesting structural or functional importance. The analysis supported molecular modeling studies in suggesting several residues in positions that were consistent with ligand-binding function. Two of these positions, dominated by histidine residues, may play important roles in ligand binding and could confer broad specificity to mammalian odor receptors. The presence of positive (overdominant) selection at some of the identified positions provides additional evidence for roles in ligand binding. Higher-order groups of correlated residues were also observed. Each group may interact with an individual ligand determinant, and combinations of these groups may provide a multi-dimensional mechanism for receptor diversity.

Amino Acid Sequence↗
PubMed1994

Cloning and sequence analysis of the gene encoding human lymphocyte prolyl endopeptidase.

The human cDNA encoding prolyl endopeptidase, a cytoplasmic endoprotease which hydrolyses the peptide bond at the C-terminal side of proline, was sequenced. After the isolation of the 3' terminal fragment of the pep cDNA sequence from a human lymphocyte cDNA library, an approach based on the polymerase chain reaction (PCR) was undertaken to obtain the complete pep cDNA. Overlapping DNA fragments were generated by PCR from cDNA synthesized from human lymphocyte mRNA. The DNA fragments were subcloned and sequenced. The complete cDNA is 2562 nucleotides (nt) in length and contains an open reading frame coding for a protein of 710 amino acids (aa). Comparison of the primary PEP sequences from human lymphocyte and pig brain shows 97% identify. The aa sequence analysis shows homology with bacterial PEPs and with protease II from Escherichia coli. Asp641 probably participates in the active site of PEP.

Amino Acid Sequence↗
PubMed1994

Protein stabilization by hydrophobic interactions at the surface.

The contribution of the solvent-exposed residue 63 to thermal stability of the thermolysin-like neutral protease of Bacillus stearothermophilus was studied by analyzing the effect of twelve different amino acid substitutions at this position. The thermal stability of the enzyme was increased considerably by introducing Arg, Lys or bulky hydrophobic amino acids. In general, the effects of the mutations showed that hydrophobic contacts in this surface-located region of the protein are a major determinant of thermal stability. This observation contrasts with general concepts concerning the contribution of surface-located residues and surface hydrophobicity to protein stability and indicates new ways for protein stabilization by site-directed mutagenesis.

Bacterial Proteins↗
PubMed1994

The role of electrostatic charge in the membrane insertion of colicin A. Calculation and mutation.

The bacterial toxin colicin A binds spontaneously to the surfaces of negatively charged membranes. The surface-bound toxin must subsequently, however, become an acidic 'molten globule' before it can fully insert into the lipid bilayer. Clearly, electrostatic interactions must play a significant role in both events. The electrostatic field around the toxin in solution was calculated using the finite-difference Poisson-Boltzmann method of the Delphi programme and the known X-ray structure. A large positively charged surface was identified which could be involved in the binding of colicin to negatively charged membranes. The applicability of the result was tested by also calculating the fields around modelled structures of the closely related colicins B and N. Surprisingly, colicin N showed a similar charge distribution in spite of its isoelectric point of pI 10.20 (colicin A has pI 5.44). One reason for this is the strong conservation of certain negative charges in all colicins. There is a single highly conserved aspartate residue (Asp78) on the positively charged face which provides a small but discrete region of negative charge. This residue, Asp78, was replaced by asparagine in the mutant D78N. D78N binds faster to negatively charged vesicles but inserts only half as fast as the wild-type protein into the membrane core. This indicates that, first, the initial membrane binding has a significant electrostatic component and, second, that the isolated charge on Asp78 plays a role in the formation of the insertion intermediate.

Amino Acid Sequence↗
PubMed1994

A novel search method for protein sequence--structure relations using property profiles.

In protein engineering and design it is very important that residues can be inspected in their specific environment. A standard relational database system cannot serve this purpose adequately because it cannot handle relations between individual residues. With SCAN3D we introduce a new database system for integrated sequence and structure analysis of proteins. It uses the relational paradigm wherever possible. Its main power, however, stems from the ability to retrieve stretches of consecutive residues with certain properties by comparing a property profile with all stretches of residues in the database, exploiting the ordered character of proteins. In doing so, it bypasses the large number of join operations that would be required by relational database systems. An additional advantage of using property profile matching is that searches can be carried out allowing a pre-set number of mismatches. Also, as the database is read-only, SCAN3D does not need interactive data update mechanisms. Queries typical of a molecular engineering environment are demonstrated with specific examples: analysis of peptides that induce local structure, analysis of site-dependent rotamers and residue--residue contact analysis.

Amino Acid Sequence↗
PubMed1994

Predicting local structural changes that result from point mutations.

Point mutations are frequently used to explore the structure and/or function of proteins. The ability to predict the structural effects of point mutations would make the planning of such experiments more reliable. We have now derived a set of detailed predictive rules based on the comparison of crystal structures of point mutants and wild types in 83 cases. Despite the surprising simplicity of these rules, they describe well the conformational changes in 85% of all point mutant structures available at present.

Amino Acid Sequence↗
PubMed1994

The effect of engineering surface loops on the thermal stability of Bacillus subtilis neutral protease.

Using genetic techniques the contribution of surface loops to the thermal stability of Bacillus subtilis neutral protease (NP-sub) was studied. Mutations were designed to make the surface of NP-sub more similar to the surface of more thermostable neutral proteases such as thermolysin (TLN). The mutations included the replacement of an irregular loop by a shorter variant and the introduction of a ten-residue beta-hairpin. In general, these drastic mutations had little effect on the production and activity of NP-sub, indicating the feasibility of major structural rearrangements at the surface of proteins. In the most stable mutant, exhibiting an increase in thermal stability of 1.1 degree C, approximately 10% of the surface of NP-sub was modified. Several NP-sub variants carrying multiple mutations were constructed. Non-additive effects on thermal stability were observed, which were interpreted on the basis of a model for thermal inactivation, that emphasizes the importance of local unfolding processes for thermal stability.

Amino Acid Sequence↗
PubMed1994

The N-terminal domain of VirG of Agrobacterium tumefaciens: modelling and analysis of mutant phenotypes.

Fourteen mutants in the N-terminal domain of virulence factor G (VirG) were obtained by random mutagenesis. Two mutants showed an altered phenotype, all others were non-functional. All mutants can still be phosphorylated and bind to DNA. A 3-D model was built based on the coordinates of chemotaxis protein Y (CheY). Many of the observed phenotypic changes of VirG are explained qualitatively. Combination of model building and biochemical information leads to the conclusion that the active sites of VirG and CheY must partly use different residues to perform the same phosphorylation and dephosphorylation reactions.

Agrobacterium tumefaciens↗
PubMed1993

Residues in the TATA-binding protein required to mediate a transcriptional response to retinoic acid in EC cells.

The eukaryotic TATA-binding protein TBP, which is required for transcription by RNA polymerase II, is tightly associated with a particular set of factors in the TFIID complex, and as such provides a target for transcriptional regulation exerted by upstream factors. An embryonic carcinoma (EC) cell-specific activity like that of the viral factor E1A has been implicated in the mediation of transactivation from the retinoic acid receptor to human TBP, but yeast TBP cannot perform this function. Using TBP mutants with an altered TATA-box-binding specificity, we show here that yeast TBP can mediate transcriptional activation in mammalian cells and that its inability to convey retinoic acid-dependent transactivation in EC cells is due to specific residues in its core region. These residues preclude a functional association with the cellular E1A-like activity. TBP is thus a target for retinoic acid-dependent transactivation in EC cells by providing a surface for interaction with the EC cell-specific E1A-like activity.

Adenovirus E1A Proteins↗
PubMed1993

Stabilization of Bacillus stearothermophilus neutral protease by introduction of prolines.

The thermostability of neutral proteases has been shown to depend on autolysis which presumably occurs in flexible regions of the protein. In an attempt to rigidify such a region in the neutral protease of Bacillus stearothermophilus, residues in the solvent-exposed 63-69 loop were replaced by proline. The mutations caused large positive (Ser-65-->Pro, Ala-69-->Pro) or negative (Thr-63-->Pro, Tyr-66-->Pro) changes in thermostability, which were explained on the basis of molecular modelling of the mutant proteins. The data show that the introduction of prolines at carefully selected positions in the protein can be a powerful method for stabilization.

Bacterial Proteins↗
PubMed1993

Prediction and analysis of structure, stability and unfolding of thermolysin-like proteases.

Bacillus neutral proteases (NPs) form a group of well-characterized homologous enzymes, that exhibit large differences in thermostability. The three-dimensional (3D) structures of several of these enzymes have been modelled on the basis of the crystal structures of the NPs of B. thermoproteolyticus (thermolysin) and B. cereus. Several new techniques have been developed to improve the model-building procedures. Also a 'model-building by mutagenesis' strategy was used, in which mutants were designed just to shed light on parts of the structures that were particularly hard to model. The NP models have been used for the prediction of site-directed mutations aimed at improving the thermostability of the enzymes. Predictions were made using several novel computational techniques, such as position-specific rotamer searching, packing quality analysis and property-profile database searches. Many stabilizing mutations were predicted and produced: improvement of hydrogen bonding, exclusion of buried water molecules, capping helices, improvement of hydrophobic interactions and entropic stabilization have been applied successfully. At elevated temperatures NPs are irreversibly inactivated as a result of autolysis. It has been shown that this denaturation process is independent of the protease activity and concentration and that the inactivation follows first-order kinetics. From this it has been conjectured that local unfolding of (surface) loops, which renders the protein susceptible to autolysis, is the rate-limiting step. Despite the particular nature of the thermal denaturation process, normal rules for protein stability can be applied to NPs. However, rather than stabilizing the whole protein against global unfolding, only a small region has to be protected against local unfolding. In contrast to proteins in general, mutational effects in proteases are not additive and their magnitude is strongly dependent on the location of the mutation. Mutations that alter the stability of the NP by a large amount are located in a relatively weak region (or more precisely, they affect a local unfolding pathway with a relatively low free energy of activation). One weak region, that is supposedly important in the early steps of NP unfolding, has been determined in the NP of B. stearothermophilus. After eliminating this weakest link a drastic increase in thermostability was observed and the search for the second-weakest link, or the second-lowest energy local unfolding pathway is now in progress. Hopefully, this approach can be used to unravel the entire early phase of unfolding.

Amino Acid Sequence↗
PubMed1993

Modeling of transmembrane seven helix bundles.

Transmembrane seven helix bundles form a large family of membrane inserted receptors and are responsible for a wide range of biological functions. Experimental data suggest that their overall structure is similar to bacteriorhodopsin. We describe here a new approach for the modeling of transmembrane seven helix bundles based on statistically derived environmental preference parameters combined with experimentally determined features of the receptors. The method was used to create a model for the human beta 2-adrenoreceptor. This model is physically plausible, is in reasonable agreement with experimental data and may be helpful in planning new receptor engineering experiments.

Amino Acid Sequence↗
PubMed1993

Molecular characterization of an extracellular acid-resistant lipase produced by Rhizopus javanicus.

An extracellular lipase (triacylglycerol acylhydrolase EC 3.1.1.3), produced by the fungus Rhizopus javanicus was purified to homogeneity using an expeditious two-step isolation method. The enzyme, with a molecular mass of 36 kDa and a specific activity of 9260 microequivalent of fatty acid released per minute and mg under standard conditions, consists of three isoforms with isoelectric points of 7.8, 7.7, and 7.1, respectively. The purified lipase was digested using chemical and enzymatical procedures: CNBr cleavage, partial acid hydrolysis, and proteolytic cleavage by means of trypsin. Amino-acid sequencing of the resulting peptides indicates that the three lipases from Rhizopus javanicus, Rhizopus niveus and Rhizopus delemar are produced as identical proenzymes but processed differently. These Rhizopus lipases show 54% identity with the lipase from Rhizomucor miehei. Using the structure of the Rhizomucor miehei lipase, the molecular model of Rhizopus javanicus lipase was constructed. Both enzymes are alpha/beta type proteins with a central 8-stranded mixed beta-pleated sheet and have a remarkably similar distribution of hydrophobic amino acids at their surface. The tryptophan in the center of the helical lid covering the active site of Rhizomucor miehei lipase is mutated into an alanine, indicating that it is not essential for the proper movement of the helical lid.

Amino Acid Sequence↗
PubMed1993

On the specificity of carboxypeptidase N, a comparative study.

The structure of the enzymatically active subunit of human plasma carboxypeptidase N was modeled based on the homology with bovine carboxypeptidase A. The active site of carboxypeptidase N is well conserved in comparison with carboxypeptidase A. From a comparison of energetically favorable binding sites for different atomic probe groups a hypothesis for the differences in substrate specificity between carboxypeptidases A and N was derived. Small synthetic peptide substrates were synthesized to confirm this hypothesis. This study shows that even with very low homology model building by homology can be employed to build models of sufficient quality to aid in drug design.

Amino Acid Sequence↗
PubMed1992

Increasing the thermostability of the neutral proteinase of Bacillus stearothermophilus by improvement of internal hydrogen-bonding.

In an attempt to increase the thermostability of the neutral proteinase of Bacillus stearothermophilus the buried Ala-170 was replaced by serine. Molecular-dynamics simulations showed that Ser-170 stabilizes the enzyme by formation of an internal hydrogen bond. In addition, the hydroxy group of Ser-170 could contribute to stability by filling an internal cavity. After the introduction of the mutation, using site-directed-mutagenesis techniques, an increase in stability of 0.7 +/- 0.1 degrees C was obtained.

Alanine↗