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I Lasters

Publications and source records attributed to I Lasters.

At least 37 records · Page 2Linked to original sources
PubMed1991

Two structural domains as a general fold of the toxic fragment of the Bacillus thuringiensis delta-endotoxins.

The unfolding by guanidine hydrochloride of the toxic fragment of a Bacillus thuringiensis toxin belonging to the CryIC class reveals a two-step denaturation under both acid and alkaline conditions. This demonstrates the existence of two structural domains as building blocks for this toxin. Protease digests performed on a CryIA(b) and CryIC B. thuringiensis toxin, under native and partially denatured conditions, confirm this conclusion. Whereas the native CryIC toxin is completely protease resistant, the CryIA(b) toxin, earlier described as consisting of two structural domains [Convents, D., Houssier, C., Lasters, I. & Lauwereys, M. (1990) J. Biol. Chem. 265, 1369-1375], is cleaved by three proteases, resulting in at least two common fragments. This suggests that this toxin is built up of two globular units linked by a protease-susceptible linker. The detection of a stable intermediate along the denaturation curve allows us to study and compare the consecutive unfolding of the structural domains for both toxins. By addition of a protease, under conditions where such an unfolding intermediate exists, a single denaturation phase can be assigned to a specific part of the protein. These experiments lead to the conclusion that the domain whose stability is highly dependent on pH corresponds to the N-terminal half of both toxins.

Amino Acid Sequence↗
PubMed1991

A model for histone H5-DNA interaction: simultaneous minor and major groove binding.

Using the tertiary structure of the globular domain of H5 (GH5) and based on an alternative sequence homology between GH5 and DNA-binding proteins containing the helix-turn-helix motif, a model for H5-DNA interaction is proposed. From molecular graphics it follows that helix II recognizes the major groove of the DNA, as does the second helix of the helix-turn-helix motif, while helix III makes minor groove contacts, in agreement with the hypothesis of Turnell et al. (FEBS letters 232, 263-268). In the resulting model GH5 makes contact with a full turn of DNA.

Amino Acid Sequence↗
PubMed1991

Enhancing the thermostability of glucose isomerase by protein engineering.

We have engineered recombinant glucose isomerase (GI) from Actinoplanes missouriensis by site-directed mutagenesis to enhance its thermal stability in both the soluble and immobilized forms. Substitution of arginine for lysine at position 253, which lies at the dimer/dimer interface of the GI tetramer, produced the largest stabilization under model industrial conditions. We discuss our results in terms of a model in which chemical glycation of lysines by sugars in the industrial corn syrup substrate represents a major pathway of destabilization.

Aldose-Ketose Isomerases↗
PubMed1990

The Bacillus thuringiensis delta-endotoxin. Evidence for a two domain structure of the minimal toxic fragment.

The conformational characteristics of the minimal toxic fragment of the delta-endotoxin from Bacillus thuringiensis berliner 1715 were examined by fluorescence and circular dichroism spectroscopy. This insecticidal protein, specifically toxic to lepidopteran species, was found to consist of two structural domains. Experimental evidence for this conclusion was provided by biphasic guanidine hydrochloride unfolding curves at different pH values and electrophoretic patterns of protease digests. Two stable fragments of comparable molecular weight were obtained using four different broad specificity proteolytic enzymes. A secondary structure model was constructed using seven B. thuringiensis toxin sequences. These toxins were selected on the basis of their limited sequence homology and represent all known insecticidal specificities. Despite this divergence, a consensus secondary structure pattern was obtained, confirming the structural homology among the toxins. The N-terminal halves of all toxins are predicted to be relatively rich in alpha-helix structure and the C-terminal parts to contain alternating beta-strand and coil structures. The latter seems characteristic for a beta-sheet conformation. Comparing this model to the unfolding data obtained by circular dichroism, whose far UV signal gives a measure of the alpha-helix content, allowed us to delineate the structural domains into the primary structure.

Amino Acid Sequence↗
PubMed1990

The design of idealized alpha/beta-barrels: analysis of beta-sheet closure requirements.

The 8-fold parallel alpha/beta-barrel topology is encountered in proteins that display an impressive variety of functions, suggesting that this topology may be a rather nonspecific and stable folding motif. Consequently, this motif can be considered as an interesting framework to design novel proteins. It has been shown that the shape of the beta-sheet portion of the barrel can be approximated by a hyperboloid. This geometric object may therefore be used as a scaffold to construct an idealized eight-stranded beta-barrel. To facilitate the de novo design of such structures, a collection of modeling tools has been developed allowing secondary structure elements to be mapped onto the scaffold surface and rotation and translation operations to be performed about user defined axes while evaluating their contribution to the conformational energy of the system. These tools have been applied in a systematic study assessing the phi, psi requirements to design symmetric eight stranded beta barrels with optimal hydrogen bonding between adjacent beta-strands. It is observed that: (a) the beta-sheet structure can be closed without introducing irregular stagger between beta-strands and (b) the region of phi, psi dihedral angle space compatible with the formation of regular symmetric eight stranded beta-barrels coincides with the phi, psi region corresponding to average beta-strands in known protein structures, suggesting that barrel closure does not impose gross constraints on beta-strand geometry.

Computer Simulation↗
PubMed1990

Estimating the twist of beta-strands embedded within a regular parallel beta-barrel structure.

The parallel beta-barrel is a recurrent structural motif found in a large variety of different enzymes belonging to the family of alpha/beta-proteins. It has been shown previously that the hyperboloid can be considered as a scaffold describing the parallel beta-barrel structure. To assess restraints on beta-strand twist imposed by a given scaffold geometry, the notion of scaffold twist, Ts, is introduced. From Ts, the beta-strand twist (Tw beta) expected for a given scaffold geometry can be derived and it is verified that this computed twist can be used to identify beta-barrels characterized by good hydrogen bonding. It is noted that Tw beta is only slightly affected for beta-barrels differing in the number (N) of beta-strands, suggesting that restraints on main-chain conformation of beta-strands are not likely to account for the N = 8 invariability observed in natural parallel beta-barrels thereby strengthening previous work rationalizing this constancy.

Enzymes↗
PubMed1990

Basic design features of the parallel alpha beta barrel, a ubiquitous protein-folding motif.

Basic design features of the beta-sheet portion in parallel alpha beta barrels in known protein structures are analysed in the context of a model of a regular hyperboloid. A formal description of the relationships between beta-sheet twist, number of strands in the sheet and barrel dimensions is derived, and the underlying physical principles are rationalized. Results suggest that the major constraints on the geometry of the beta-sheet portion of the barrel come from the requirements to have optimal H-bonding interactions between beta-strands and to closely pack amino acid side-chains in the barrel interior so as to exclude bulk water. In addition, we show how the hyperboloid model and the ensuing formalism can serve to derive useful geometric and graphic tools for computer-aided protein design de novo. We then illustrate how these tools are used to determine that the requirement to have a closed regular eight-stranded beta-sheet surface imposes no particular constraints on the geometry (phi, psi angles) of the polypeptide backbone. Understanding the role of the amino acid sequence in determining the observed structures remains a major challenge. Detailed comparisons of known alpha beta-barrel structures (and amino acid sequence) with each other, and with polypeptide fragments from other protein crystal structures, reveal only a limited number of common sequence-structure motifs. These belong to characteristic alpha beta 1 and alpha beta 3 loop families previously described in alpha beta proteins, and occur at least once in nearly all the alpha beta-barrel structures examined.

Amino Acid Sequence↗
PubMed1989

Modelling the polypeptide backbone with 'spare parts' from known protein structures.

An automatic procedure for building a protein polyalanine backbone from C alpha positions and 'spare parts' retrieved from a data base of 66 high-resolution protein structures is described. Protein backbones are constructed from overlapping fragments of variable length, which allows the backbone of regular secondary structure elements to be built in one block. The procedure is shown to yield backbones which compare very favourably with those from highly refined X-ray structures (r.m.s. deviation between generated and crystal structures less than 1A). The method is furthermore quite insensitive to experimental errors in C alpha positions as well as to the size of the data base, and is seen to yield valuable insight into the relationships between sequence and 3-D structure: one example on triose phosphate isomerase, a beta-barrel protein, shows that beta alpha loops can be considered as structurally more uncommon than alpha beta loops. The 'spare parts' approach is also found to be useful for general-purpose modelling of local structural changes produced by insertion or deletion of residues. It should, however, be used with caution. Crude selection criteria based solely on fragment length and geometric fit to the loop base regions yield realistic backbones in about two-thirds of the test cases (r.m.s. deviations from refined crystal structure approximately 1A). In the remaining cases, sequence information, in particular the presence of glycine residues which tend to adopt more unusual backbone conformations, must be considered to obtain comparable results.

Carboxypeptidases↗
PubMed1988

Structural analysis of the 2.8 A model of Xylose isomerase from Actinoplanes missouriensis.

The structure of Xylose isomerase (X.I.) from Actinoplanes missouriensis has been solved to 2.8 Angstroms resolution. Phases were determined from a single Eu3+ derivative and from the noncrystallographic 222 symmetry of the tetrameric molecule. An atomic model was built and subjected to restrained crystallographic refinement. The resulting model is shown to be closely similar to the recently reported X.I.'s structures from three other bacterial sources. Each monomer is found to be composed of an eight-stranded alpha/beta "T.I.M." barrel forming an N-terminal domain of 328 residues followed by a large loop of 66 residues embracing an adjacent subunit. Analysis of intersubunit packing shows that the X.I. tetramer is an assembly of two tight dimers. The beta barrel fits a simple hyperboloid model as other T.I.M. barrels do. The active site, identified as the binding site for the inhibitor xylitol, is located at the carboxyl end of the beta strands in the barrel next to a pair of binding sites for Eu3+ ions, which are assumed to be sites for the divalent ions involved in catalysis. Active sites in the tetramer are oriented towards the interface between dimers. It is suggested that subunit interfaces might stabilize the active site region and this might explain the oligomeric nature of other alpha/beta barrel enzymes.

Actinomycetales↗
PubMed1988

Structural principles of parallel beta-barrels in proteins.

Eight-stranded beta-sheets in nine protein structures containing "TIM (triose phosphate isomerase) barrels" are shown to be fitted satisfactorily by hyperboloids, the generating lines of which pass through the beta-strands. Simple parameterizations of the hyperboloid model are then used to determine the constraints that govern key parameters, such as the number of strands in the barrel, and to rationalize the remarkable conservation of strand number, observed to be eight, in nearly all the known examples of parallel beta-barrels. It is shown that the requirement to exclude solvent from the barrel interior, while at the same time keeping an upper limit on strand twist and interstrand distance so as to foster extensive hydrogen bonding interactions within the sheet, imposes strong constraints on barrel geometry. A formal description of the relationships between beta-sheet twist, strand number, and barrel dimensions is given here. It could have important implications for studies of protein folding and design.

Computer Simulation↗
PubMed1985

Scatter analysis of discrete-sized chromatin fragments favours a cylindrical organization.

Fragments of chromatin containing 23 +/- 2.5 nucleosomes have been fractionated after light nuclease treatment of chicken erythrocyte nuclei. Low-angle scattering measures the total z-average radius of gyration of the already well-defined particles and the shape of scatter curves can be compared with three-dimensional analysis as opposed to cross-section analysis of long chromatin fragments. The data show that the particles are not spherical, have no detectable hole in the center of the structure and are best represented by a solid rod-like shape such as that generated by a coil of nucleosomes with the centre perhaps filled with linker DNA and histone H1/H5. 23 nucleosome fragments, where the DNA is partially fragmented, have near-identical scatter curves to the above-defined intact particles, indicating the primary importance of histone proteins in maintaining the integrity of the chromatin higher-order structure. Neutron scattering shows the radii of gyration to be contrast-independent, which fits in with the model calculations for solenoids. Particles with fragmented DNA and the intact particles, therefore, behave as sections of a solenoidal higher-order structure and possibly are observed as "superbeads' only during the folding and unfolding pathways of nucleosome multimers.

Animals↗
PubMed1985

Assembly of oligonucleosomes into a limit series of multimeric higher-order chromatin structures.

Chicken erythrocyte chromatin, obtained after fragmentation with micrococcal nuclease, appears to remain folded in a stable distribution of supranucleosomal structures in buffers containing 80 mM NaCl. These supranucleosomal particles are composed of on average 25 nucleosomes. However, the integrity of the linker DNA within these particles is not required. The supranucleosomal particles have been interpreted by others as superbeads cut out of a preexisting granular nominal 30-nm chromatin fibre. We show that the same distribution of supranucleosomal structures (even those containing internal DNA scissions) can be reconstituted from unfolded nuclear chromatin extracts as present in 10 mM or 600 mM NaCl. Moreover, fractions of oligonucleosomes with mean lengths between 6 and 15 nucleosomes reassemble or aggregate into a limit series of multimeric species. The existence of an assembly barrier could be inferred as we were unable to observe a stable and soluble assembly product containing more than about 25 nucleosomes. We propose an alternative explanation for the generation and observation of a constant distribution of supranucleosomal structures in nuclear extracts, based on the assembly or aggregation property of oligonucleosomes and on the existence of an assembly barrier.

Animals↗
PubMed1981

The structural organization of dinucleosomes and oligonucleosomes. Electric dichroism and birefringence study.

The spatial organization of nucleosomes and linker DNA in dinucleosomes and oligonucleosomes of various chain lengths has been investigated through electric dichroism, birefringence and relaxation times measurements at low ionic strengths (0.5 to 2.2 mM). From the negative dichroism observed for all the samples, it is concluded that the nucleosome subunits in the oligonucleosome chain must lie with their disc planes closely parallel to the fibre axis. The large increase of the negative dichroism of dinucleosomes upon Hl removal is interpreted by the unwinding of the DNA tails and the internucleosomal segment. All the samples displayed, under bipolar pulses, a predominantly induced orientation mechanism.

Animals↗
PubMed1981

Protection of discrete DNA fragments by the complex H1-octamerhistones or H5-octamerhistones after micrococcal nuclease digestion.

Several authors, including ourselves, have reported the existence of chromatosomes with DNA size larger than 166 bp in bird erythrocyte chromatin. It was tempting to correlate this increased DNA size with the presence of histone H5. In order to substantiate this hypothesis, we performed a micrococcal nuclease digestion kinetic on: chicken erythrocyte chromatin, either native, selectively depleted from H1, or from H1 and H5; and rat liver chromatin, either native or partially H1 depleted. The comparative analysis of the lengths of DNA in the chromatosome size region led to the following conclusions: - denaturing gels clearly reveal a first discrete pause at 178 nucleotides in H1 depleted chicken erythrocyte chromatin as well as in partially H1-depleted rat liver chromatin, before the material accumulates at the next intermediate 166 nucleotide chromatosome pause. - the generation of all discrete chromatosome bands is critically dependent on low ionic strength conditions and low Ca++ concentrations during the digestion, suggesting it may result from the protection of DNA cleavage sites by histone H5 or H1, C or N terminal domains.

Animals↗
PubMed1981

Differences in rearrangements of H1 and H5 in chicken erythrocyte chromatin.

H1 can rearrange in chicken erythrocyte polynucleosomes in 80 mM NaCl buffers. These rearrangements have been studied by sedimentation analysis. H1 redistributes between polynucleosomes as well as between polynucleosomes and monosomes. In these rearrangements H1 molecules move to free DNA sites. In contrast to H1, the chicken erythrocyte specific lysine-rich histone H5 does not show any of these dynamic properties. This difference in mobility of H1 and H5 also manifests itself in the selective extraction of H1 from H1, H5 containing polynucleosomes by the cation-exchange resin AG 50W-X2 at 80 mM NaCl.

Animals↗
PubMed1980

Upon the observation of superbeads in chromatin.

There exist some indications that nucleases recognize "superbeads" in chromatin. We show that a chromatin extract of rat liver which contains so-called "superbead"-peaks can be separated in a Mg++ soluble and a Mg++ insoluble fraction. The Mg++ insoluble fraction contains the full complement of histones and the expected DNA fragments, but has lost the characteristic peaks in sucrosegradient profiles. These discrete peaks are found in the Mg2+ soluble fraction of the chromatin extract. We give evidence that these peaks are RNP particles on the basis of their protein- and nucleic acid contents.

Animals↗