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J Haiech

Publications and source records attributed to J Haiech.

At least 37 records · Page 2Linked to original sources
PubMed1995

Use of TSAR as a new tool to analyze the molecular dynamics trajectories of proteins.

There is a lack of tools to analyze simulations of protein molecular dynamics quantitatively. Our aim is to use calmodulin, a prototypical calcium-binding protein, to describe a strategy and some tools for extracting relevant information from dynamics calculations. Our main conclusions are as follows: Autocorrelation vectors may be used to represent a 3D conformation in an n-dimensional space, where n is variable (n < or = 20-30). On such a transformation, classic statistical tools (PCA, clustering, etc.) may be used to differentiate or characterize dynamics trajectories quantitatively. TSAR, an integrated package used for quantitative structure-activity relationships, is well suited (after minor modifications) for such a purpose. Finally, this type of strategy is able to point out the effects of the solvent screening parameters of the Amber software on the dynamics trajectories of calmodulin.

Calmodulin↗
PubMed1995

Analysis of errors in finished DNA sequences: the surfactin operon of Bacillus subtilis as an example.

Increased productivity in DNA sequencing would not be valid without a straightforward detection and estimation of errors in finished sequences. The sequence of the surfactin operon from Bacillus subtilis was obtained by two different groups and by chance we were also working on the same chromosome region. Taking advantage of this situation we report in this paper, the number and nature of errors found in the overlapping part of the DNA sequences obtained by the three laboratories. The coincidence of some of the errors with compression in sequence ladders and with secondary DNA structures as well as the detection of frameshift errors using computer programs, are demonstrated. Finally we discuss the definition of a new sequencing strategy that might minimize both the error rate and the cost of sequencing.

Artifacts↗
PubMed1994

Investigating the high affinity and low sequence specificity of calmodulin binding to its targets.

Calmodulin (CaM) is a calcium binding protein that regulates a wide range of enzymes. Recently the structures of a number of complexes between CaM and synthetic target peptides have been determined. The peptides correspond to the CaM-binding domain of skeletal and smooth muscle myosin light-chain kinase (MLCK) and calmodulin-dependent protein kinase II alpha. Comparison of the peptide-free and peptide-bound structures reveals that CaM undergoes a large conformational change when forming a complex, resulting in the formation of a binding surface that provides for an optimal interaction with its target. In this work, the available co-ordinates of the NMR solution structure of CaM-skeletal MLCK peptide are used as a basis upon which several molecular models of binding are built. The detailed features of the protein's peptide binding surface are revealed through two-dimensional topographical projections. Negatively charged margins at the binding surface extremities interact strongly with basic peptide residues separated by nine or ten positions. The binding surface core is hydrophobic and displays a groove with four deep pockets, which can accommodate bulky peptide residues at relative positions 4 and 8 (pocket A), 11 (pocket B), 13 (pocket C), 14 and 17 (pocket D). Therefore, both electrostatic and van der Waals' features contribute to the high affinity binding. A search for alternative peptide placements in the binding tunnel reveals the dominant role of specific electrostatic interactions in the binding energy. Apolar interactions are more permissive, such that the hydrophobic side-chains that line the binding tunnel adapt in order to maintain favourable van der Waals' contacts. The model suggests that the structure can accommodate large peptide translations (up to 5 A) and a reversed peptide binding mode, with a little loss in binding interaction energy. These calculations are compared with available experimental data, providing a structural rationale for the low sequence specificity of the CaM target recognition.

Amino Acid Sequence↗
PubMed1994

Gain of function mutations for yeast calmodulin and calcium dependent regulation of protein kinase activity.

Yeast calmodulin binds only three calcium ions in the presence of millimolar concentrations of magnesium due to a defective calcium-binding sequence in its carboxyl terminal domain. Yeast calmodulin's diminished calcium-binding activity can be restored to that of other calmodulins by the use of site-directed mutagenesis to substitute its fourth calcium-binding domain with that of a vertebrate calmodulin sequence. However, the repair of yeast calmodulin's calcium-binding activity is not sufficient to repair quantitatively yeast calmodulin's defective protein kinase activator activity. Yeast calmodulin's activator activity with smooth muscle and skeletal muscle myosin light chain kinases and brain calmodulin-dependent protein kinase II can be progressively repaired by additional substitutions of vertebrate calmodulin sequences, provided that the four calcium-binding sites remain intact. An unexpected result obtained during the course of these studies was the observation that myosin light chain kinases from smooth and skeletal muscle tissues can respond differently to mutations in calmodulin. These and previous results indicate that the binding of four calcium ions by calmodulin is necessary but not sufficient to bring about quantitative activation of protein kinases, and are consistent with the conformational selection/restriction model of the dynamic equilibrium among calcium, calmodulin and each calmodulin regulated enzyme.

Amino Acid Sequence↗
PubMed1994

A protein/peptide assay using peptide-resin adduct: application to the calmodulin/RS20 complex.

To obtain equilibrium and kinetic constants of a protein/peptide complex, we have developed a rapid procedure which uses peptides specifically linked to a resin. With this peptide-resin adduct, bound and free 125I-labeled protein could be easily separated by simple centrifugation. The feasibility of the method was demonstrated with the calmodulin/RS20 complex, where RS20 is the putative calmodulin binding peptide of the smooth muscle myosin light chain kinase (smMLCK). In addition to the wild-type calmodulin (SYNCAM) expressed in Escherichia coli, we also examined calmodulin mutants with charge reversals called SYNCAM12A (DEE 118-120-->KKK) and SYNCAM18A (EEE 82-84-->KKK and DEE 118-120-->KKK). The kinetic analysis of the interaction between SYNCAM and RS20 associated with titration experiments allowed us to measure dissociation constants (KD) in the range of 10(-9) M, in good agreement with previously published data. Moreover, the binding assays showed that SYN-CAM18A did not interact with RS20, whereas SYN-CAM12A did with a KD around 10(-8) M. The lack of binding of SYNCAM18A to RS20 provides an explanation for the lack of smMLCK activation by SYNCAM18A. Altogether, these results demonstrate that peptide-resin can be used as a tool for separating bound from free protein, thus enabling a rapid and reliable quantification of the protein/peptide interaction.

Amino Acid Sequence↗
PubMed1992

Purification of assembly-competent tubulin from Saccharomyces cerevisiae.

We have developed a straightforward, two-step procedure to isolate highly purified yeast tubulin that reproducibly assembles into microtubules. The starting extracts are obtained from cells genetically engineered to overproduce both the alpha and beta subunits of tubulin, under control of the galactose promoter, to approximately 10-times wild-type levels. The first step of purification is carried out with the high-speed supernatant of lysed cells loaded onto a DEAE-Sephadex column; after this step the tubulin preparation is approximately 30% pure. In the second step, the tubulin fractions are loaded onto an immunoaffinity column prepared by coupling the anti-(alpha-tubulin) monoclonal antibody YL 1/2 to Sepharose-4B. Following elution with 0.8 M KCl, the tubulin present in the peak is 90% pure. Upon addition of porcine brain microtubule-associated proteins or DEAE-dextran, this tubulin preparation is functionally active for assembly into microtubules, as visualized by electron microscopy on negatively stained samples. Virtually identical microtubule structures are produced in parallel experiments on the assembly of yeast or porcine brain tubulin, with differences observed only at acidic pH values. Overall, this relatively simple procedure provides a useful tool for the production of functional tubulin suitable both for structural studies and for investigations of the assembly process.

Animals↗
PubMed1992

The heterodimer calmodulin: myosin light-chain kinase as a prototype vertebrate calcium signal transduction complex.

The heterodimer complex of calmodulin (CaM) and the protein kinase catalytic subunit of myosin light chain kinase from vertebrate smooth muscle and non-muscle tissues (sm/nmMLCK) is one of the most extensively characterized CaM-regulated enzyme complexes and it has an established in vivo role in the transduction of calcium signals into biological responses. We have used a combination of approaches to the study of CaM and sm/nmMLCK in order to derive initial insight into the key features of each protein and of the CaM-MLCK heterodimeric complex that are involved in protein-protein and calcium-protein recognition and regulation of enzyme activity. On-going studies are described here that include site-specific mutagenesis, fluorescence spectroscopy, enzymology and peptide analog analysis. These and previous results indicate that: (1), both electrostatic and hydrophobic features are important in the functionally correct interactions between CaM and MLCK; (2), even the interactions between CaM and peptide analogs of the CaM binding site of MLCK are heterogeneous and non-trivial in nature; (3), amino-acid residues that have been conserved in CaM across millions of years of evolution and that are conserved in CaMs with quantitative MLCK activator activity can be mutated without any detectable effect on activity and (4), structures different from the prototypical EF-hand domain of CaM can have similar calcium-binding activity in the presence of a CaM binding structure.

Amino Acid Sequence↗
PubMed1992

Ca(2+)-calmodulin regulated effectors of microtubule stability in bovine brain.

Stable microtubules (as defined by resistance to Ca2+, drug or cold temperature induced disassembly) form in abundance during tubulin assembly in brain crude extracts. We have previously shown that, in rat brain crude extracts, all microtubule stabilizing activity could be ascribed to a single Ca(2+)-calmodulin binding and Ca(2+)-calmodulin regulated protein, called "stable tubule only polypeptide", STOP145 [Pirollet, F., Rauch, C. T., Job, D., & Margolis, R. L. (1989) Biochemistry 28, 835-842]. We have now performed an exhaustive study of STOP-like effectors in bovine brain high-speed supernatants. All activity binds to cation exchangers and to Ca(2+)-calmodulin affinity columns. The activity can be resolved into two peaks on sizing columns. The first eluted peak contains a prominent 220-kDa protein. The second peak contains an apparently homogeneous 20-kDa polypeptide. A monoclonal antibody specific to rat brain STOP145 recognizes the 220-kDa protein, but not the 20-kDa species. The 220-kDa protein can be purified on a STOP antibody column and accounts for the bulk of stabilizing activity in the first peak. The 20-kDa protein does not bind to STOP antibody affinity columns. Sequence analysis of oligopeptide fragments of the 20-kDa protein shows 100% homology with bovine myelin basic protein (MBP). Anti-MBP antibodies recognize the 20-kDa, but not the 220-kDa species. We conclude that the 220-kDa protein is the bovine equivalent to rat brain STOP145 and that the 20-kDa species is MBP. Microtubule stabilization by MBP and STOP220 is abolished in the presence of Ca(2+)-calmodulin, and inhibition curves are similar for both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Use of engineered proteins with internal tryptophan reporter groups and pertubation techniques to probe the mechanism of ligand-protein interactions: investigation of the mechanism of calcium binding to calmodulin.

Stopped-flow kinetic and fluorescence spectroscopic analyses, including solvent and temperature perturbations, of five isofunctional structural mutants of calmodulin indicate that calcium binding to calmodulin follows the order site III, site IV, site I, site II, with dissociation occurring in the reverse order. Each of the isofunctional structural mutants contains a single tryptophan residue, introduced by site-specific mutagenesis, as an internal spectroscopic reporter group that was used as a probe of local conformational change. Calcium binding was studied by using flow dialysis or by using fluorescence spectroscopy and monitoring the change in the single tryptophan residue in each calcium-binding site. Calcium removal was examined by using EDTA and monitoring tryptophan fluorescence or by using Quin 2 and monitoring the change in the chromophoric chelator. Computational analysis of the data suggests a rate-limiting step for dissociation between calcium removal from sites I/II and sites III/IV. Unexpected results with the site IV isofunctional mutant (Q135W-CaM) indicated cross-talk between the amino and carboxyl terminal halves of CaM during the calcium-binding mechanism. Studies with ethylene glycol provided empirical data that suggest the functional importance of the electrostatic potential of CaM, or the molarity of water, in the calcium-binding process. Altogether, the data allowed a kinetic extension of the sequential, cooperative model for calcium binding to calmodulin and provided values for additional parameters in the model of calcium binding to CaM, a prototypical member of the family of proteins required for calcium signal transduction in eukaryotic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Restoration of the calcium binding activity of mutant calmodulins toward normal by the presence of a calmodulin binding structure.

The altered calcium binding activity of calmodulins (CaM) with point mutations can be restored toward that of wild type CaMs by the formation of a complex between CaM and a CaM binding sequence. Three different site-specific mutations resulted in selective effects on the apparent stoichiometry and affinity of CaM for calcium, with maintenance of the ability to activate myosin light chain kinase. The effects on calcium binding, however, were suppressed when the mutant CaMs were complexed with RS20, a peptide analog of a myosin light chain kinase CaM binding site. The mutations included: 1) a Glu----Ala mutation at two phylogenetically conserved calcium ligands in the second (E67A-CaM) and fourth (E140A-CaM) sites; and 2) a Ser----Phe mutation at residue 101 (S101F-CaM) which affects ion channel regulation. The mutant CaMs bind 4 calciums in the absence of magnesium, but two sites have approximately 60- to 300-fold weaker binding than wild-type CaM (SYNCAM CaM). E67A-CaM and E140A-CaM bound only two calciums and S101F-CaM bound 4 calciums in the presence of magnesium. E67A-CaM and E140A-CaM recovered the ability to bind 4 calcium ions in the presence of the RS20 CaM binding peptide. These results are consistent with models in which the calcium binding activity of CaM within a supramolecular complex is different from purified CaM and raise the possibility that the selective functional effects of in vivo mutations in the calcium binding sites of CaM might be partially due to the ability of some CaM binding proteins to select and utilize CaM conformations with calcium ligation structures different from the so-called canonical EF-hand.

Amino Acid Sequence↗
PubMed1991

Cellulase EGZ of Erwinia chrysanthemi: structural organization and importance of His98 and Glu133 residues for catalysis.

Biochemical, genetic and primary sequence analyses of the Erwinia chrysanthemi endoglucanase EGZ allowed us to identify two functional domains and to locate their boundaries. The catalytic domain extends from residue 1 to 288, while a domain required for EGZ to bind to microcrystalline cellulose lies from residues 324 to 385. Each domain was found capable of functioning in the absence of the other. A region rich in Pro, Thr, and Ser residues links both domains and appeared to be susceptible to proteolytic attack. Based upon predictions derived from a method developed to compare sequences sharing a low level of similarity, e.g. hydrophobic cluster analysis (HCA), we analysed the importance of either residue His98 or Glu133 in EGZ catalytic activity. Two EGZ-derived proteins were engineered in which either His98 or Glu133 amino acid was converted to an Ala residue. Characterization of the purified proteins showed that no enzymatic activity could be detected, by using carboxymethylcellulose (CMC) or paranitrophenyl-cellobioside (pNPC) as substrates, while both mutated proteins retained the capacity to bind to microcrystalline cellulose. These studies, which to date constitute the first experimental testing of HCA-derived predictions, allowed us to identify two particular amino acids involved in cellulolytic activity. By taking into account data from chemical modification studies of other cellulases, we speculate that the His98 residue is involved in the folding of the catalytic domain while the Glu133 residue intervenes directly in the beta, 1-4 glycosidic bond cleavage.

Amino Acid Sequence↗
PubMed1990

Mutant analysis approaches to understanding calcium signal transduction through calmodulin and calmodulin regulated enzymes.

An example set of site-specific mutagenesis studies of calmodulin has been discussed in terms of strategy and how the results can provide insight into the functioning of calmodulin. A set of common examples for the study of calcium binding and enzyme activation were discussed. Essentially, site-specific mutagenesis in these initial studies is a perturbation approach. From these perturbation studies, structural features can be correlated in future studies with function and mechanisms of action proposed. More importantly, the approach allows efficient testing of proposed mechanisms and further probing of the molecular aspects of the signal transduction pathways. Clearly, the key functional feature that must be addressed in future studies is how the calcium binding steps in the mechanism are coupled to the enzyme activation step, which is the final step of the calmodulin-enzyme binding mechanism.

Amino Acid Sequence↗
PubMed1990

PCDRA: PC interactive molecular representation and modeling system.

PCDRA was designed to provide the average biologist with a user-friendly molecular display on a low-cost personal computer. The package is menu driven and is built so that a biologist, with little or no computing knowledge, finds it easy to use. The system gives a color representation with depth cueing of a protein whose atomic coordinates are stored as a PDB file. Moreover, the system presents several features similar to HYDRA and therefore is a good introduction to molecular graphics, especially for beginners in protein modeling.

Computer Graphics↗
PubMed1990

Identification of the calmodulin binding domain of alpha-fodrin and implications for folding.

A cDNA clone producing a protein that binds calmodulin has been isolated from a mouse macrophage library. The cDNA was sequenced and identified as coding for fodrin. By deleting part of the sequence, the calmodulin binding domain was located. The site is situated on repeat 11 of fodrin probably on its extra arm. This part of the sequence exhibits great similarity to other calmodulin binding proteins. Analysis of the sequence and spatial structure of calmodulin revealed a domain which is quite complementary to the sequence identified on fodrin. These results provide a new insight into the structure of fodrin and consequently into the structure of proteins of the spectrin family. A model for the general folding of these molecules is proposed, involving a simple three-layer folding. The structure was further corroborated by analysis of charge distribution in the vicinity of the calmodulin binding site. The folding we propose is in good agreement with digestion experiments and explains observations in diseases resulting from mutations of human spectrin.

Amino Acid Sequence↗
PubMed1989

Fluorescence characterization of VU-9 calmodulin, an engineered calmodulin with one tryptophan in calcium binding domain III.

Absorption and fluorescence properties of VU-9 calmodulin, an engineered calmodulin in which a tryptophan residue has been introduced in position 99, have been investigated. Tryptophan 99 fluoresces with a maximum around 348 nm and is easily quenched by fluorescence quenchers such as acrylamide, indicating that the chromophore is in a polar environment and well exposed to the solvent, a location which has been reported previously for tyrosine 99 in mammalian calmodulin [Kilhoffer, M. C., Demaille, J. G., & Gérard, D. (1981) Biochemistry 20, 4407-4414]. The quantum yields of tryptophan 99 were found to be 0.19 in the absence of calcium and 0.15 in its presence. These values indicate that the chromophore is in a particular microenvironment where it is protected from the quenching mechanisms normally occurring in proteins. Steady-state fluorescence polarization measurements indicate that the protein exhibits segmental mobility both in the absence and in the presence of calcium. Binding of calcium decreases the mobility of the chromophore, a good indication for a rigidification of the protein structure. A quite rigid structure of at least the carboxy-terminal part of VU-9 calmodulin in the presence of Ca2+ is also suggested by Förster energy-transfer measurements.

Binding Sites↗
PubMed1989

Time-resolved fluorescence study of VU-9 calmodulin, an engineered calmodulin possessing a single tryptophan residue.

An engineered calmodulin (VU-9 calmodulin), which possesses a single tryptophan residue at position 99 in calcium binding domain III, was studied by time-resolved fluorescence. At least two exponential terms are needed to describe the tryptophan fluorescence decays, either in the presence or in the absence of calcium. The characteristics of the fluorescence decays are strongly dependent upon the number of calcium ions bound per molecule of VU-9 calmodulin until half of the calcium sites are occupied, i.e., three in the absence of magnesium and two in the presence of 5 mM magnesium. A clear time-dependent spectral shift is observed in the presence of calcium. The existence of an isosbestic point in the time-resolved spectra is in agreement with a two-state model. The biexponential analysis of the 340-nm fluorescence decay during calcium titration gives parameters consistent with a two-state model in which tryptophan 99 interconverts between two different conformations, characterized by a different lifetime value, with rates altered by calcium binding. This model explains the decrease in the protein quantum yield induced by calcium binding [Kilhoffer, M. C., Roberts, D. M. Adibi, A. O., Watterson, D. M., & Haiech, J. (1989) Biochemistry (preceding paper in this issue)].

Binding Sites↗