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D M Watterson

Publications and source records attributed to D M Watterson.

At least 37 records · Page 2Linked to original sources
PubMed1994

Functional consequences in yeast of single-residue alterations in a consensus calmodulin.

A synthetic gene encoding a 'consensus' calmodulin (synCaM) was able to substitute for the Saccharomyces cerevisiae calmodulin gene (CMDI), even though synCaM is only 60% identical in primary amino acid sequence to yeast Cmd1. Twelve different synCaM mutants were also expressed in yeast. Seven of the 12 mutant synCaMs supported germination and growth of Cmd1-deficient spores. Five of the 12 mutant synCaMs were incapable of supporting germination of Cmd1-deficient spores and, of these, four were also incapable of supporting vegetative growth of Cmd1-deficient haploid cells. The five nonfunctional synCaM mutants were expressed at levels equivalent to, or higher than, the seven synCaM mutants that were able to substitute for Cmd1; thus, the inability to function was not simply due to inadequate expression or rapid degradation. All nonfunctional synCaM mutants shared a single charge reversal mutation in the central helix (E84K), which was found to be sufficient to confer the lethal phenotype. The ability of another mutant synCaM (S101F) to support growth of Cmd1-deficient cells was dependent on cell ploidy. Another mutant (K115Y) supported spore germination and vegetative growth, but not meiosis and sporulation. The terminal phenotype of cells lacking a functional calmodulin included a dramatic accumulation of polymerized microtubules.

Calmodulin↗
PubMed1993

A kinase-related protein stabilizes unphosphorylated smooth muscle myosin minifilaments in the presence of ATP.

An apparent paradox in smooth muscle biology is the ability of unphosphorylated myosin to maintain a filamentous structure in the presence of ATP in vivo, whereas unphosphorylated myosin filaments are depolymerized in vitro in the presence of ATP. This suggests that additional uncharacterized factors are required for the stabilization of myosin filaments in the presence of ATP. We report here that an abundant smooth muscle protein forms sedimentable complexes with unphosphorylated smooth muscle myosin, partially reverses the depolymerizing effect of ATP on unphosphorylated myosin, and promotes the assembly of minifilaments as revealed by electron microscopy. This protein is called kinase-related protein (KRP) because it is derived from a gene within the gene for myosin light chain kinase (MLCK) and has an amino acid sequence identical to the carboxyl-terminal domain of MLCK. Consistent with the results with purified KRP, deletion of the KRP domain within MLCK results in a diminished ability of MLCK to interact with unphosphorylated myosin. KRP binds to the heavy meromyosin fragment of myosin but not to myosin rod or fragments lacking the hinge region and light chains. Altogether, these results suggest that KRP may play a critical role in stabilizing unphosphorylated myosin filaments and that the KRP domain of MLCK may be important for subcellular targeting to filaments.

Adenosine Triphosphate↗
PubMed1993

Features of calmodulin that are important in the activation of the catalytic subunit of phosphorylase kinase.

Calmodulin (CaM) is an integral subunit, called delta, of the phosphorylase kinase hexadecamer, and the activity of the isolated catalytic gamma-subunit of the kinase is stimulated by CaM. We report here the first analysis of functionally important features of CaM for activation of the gamma-subunit. A set of genetically engineered CaMs, in which acidic residues in each of the four E-helices of the "EF-hands" were changed to basic lysine residues, was used to probe the relative importance of charge features in each domain of CaM. The maximal activation of the isolated gamma-subunit was diminished by all of the charge reversal mutations. The gamma-subunit was especially sensitive to reversals in the second and third E-helix of CaM (residues 45-47 and 82-84), the latter being present in the central helix. The results suggest the functional importance of electrostatics in the interactions between the delta-subunit (CaM) and the catalytic gamma-subunit of phosphorylase kinase, which is similar to results obtained with CaM-dependent myosin light chain kinase (MLCK) from chicken gizzard and CaM-dependent protein kinase II (CaMPK-II). However, novel features of the interaction between CaM and the gamma-subunit of phosphorylase kinase are the significant contribution of electrostatics throughout the CaM molecule, including residues in both halves and on more than one face of CaM, and the lack of a major effect of the CaM mutations on substrate kinetic parameters, unlike the effects observed with MLCK and CaMPK-II. These results are consistent with a model in which the delta-subunit (CaM) of phosphorylase kinase interacts with an extended region or multiple regions of the gamma-subunit and suggest that the mechanism of CaM activation of the gamma-subunit may have features that are distinct from those of MLCK and CaMPK-II.

Amino Acid Sequence↗
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

Inositol polyphosphate receptor and clathrin assembly protein AP-2 are related proteins that form potassium-selective ion channels in planar lipid bilayers.

We have previously described an inositol polyphosphate receptor (IPxRec), purified from detergent-solubilized bovine cerebellum microsomes, that displays potassium ion channel activity in planar lipid bilayers. We now find that the IPxRec is closely related to clathrin assembly protein AP-2. The IPxRec and AP-2 purified from bovine brain clathrin-coated vesicles share several structural and functional features: (i) similar subunit composition; each has four major polypeptides that have similar mobility (Mr values of 111,000, 100,000, 50,000, and 17,000) and relative intensity by SDS/PAGE analysis; (ii) similar size as studied by molecular sieve chromatography (Mr 400,000); (iii) identical N-terminal amino acid sequences for the Mr 50,000 subunits and Mr 111,000/100,000 doublets; (iv) immunoreactivity of the AP-2 Mr 111,000/100,000 doublet to polyclonal antibodies affinity purified against the doublet proteins of the IPxRec; (v) display of the in vitro diagnostic feature of assembly proteins--i.e., they induce the assembly of clathrin cages; and (vi) ion channel activity selective for potassium ions with the same unitary conductance when incorporated into planar lipid bilayers. One difference was found. AP-2 channels were not blocked by inositol 1,3,4,5-tetraphosphate as reported for IPx receptor channels. These studies suggest a possible connection between the IPx signaling pathways and receptor-mediated endocytosis.

Adaptor Proteins, Vesicular Transport↗
PubMed1992

Structure and expression of a calcium-binding protein gene contained within a calmodulin-regulated protein kinase gene.

We have determined the first genomic structure and characterized the mRNA and protein products of a novel vertebrate gene that encodes a calcium-binding protein with amino acid sequence identity to a protein kinase domain. The elucidation of the complete DNA sequence of this transcription unit and adjacent genomic DNA, Southern blot and polymerase chain reaction analyses of cellular genomic DNA, and examination of mRNA and protein species revealed that the calcium-binding kinase-related protein (KRP)-encoding gene is contained within the gene for a calmodulin-regulated protein kinase, myosin light-chain kinase (MLCK). The KRP gene transcription unit is composed of three exons and a 5'-flanking sequence containing a canonical TATA box motif. The TATA box, the transcription initiation site, and the first 109 nucleotides of the 5' noncoding region of the KRP mRNA correspond to an MLCK gene intron sequence. Both KRP and MLCK are produced in the same adult chicken tissue in relatively high abundance from a single contiguous stretch of genomic DNA and utilize the same reading frame and common exons to produce distinct mRNAs (2.7 and 5.5 kb, respectively) that encode proteins with dissimilar biochemical functions. There appears to be no precedent in vertebrate molecular biology for such a relationship. This may represent a mechanism whereby functional diversity can be achieved within the same vertebrate tissue by use of common exons to produce shuffled domains with identical amino acid sequences in different molecular contexts.

Amino Acid Sequence↗
PubMed1991

Fluorescence analysis of calmodulin mutants containing tryptophan: conformational changes induced by calmodulin-binding peptides from myosin light chain kinase and protein kinase II.

Peptide-induced conformational changes in five isofunctional mutants of calmodulin (CaM), each bearing a single tryptophan residue either at the seventh position of each of the four calcium-binding loops (i.e., amino acids 26, 62, 99, and 135) or in the central helix (amino acid 81) were studied by using fluorescence spectroscopy. The peptides RS20F and RS20CK correspond to CaM-binding amino acid sequence segments of either nonmuscle myosin light chain kinase (nmMLCK) or calmodulin-dependent protein kinase II (CaMPK-II), respectively. Both steady-state and time-resolved fluorescence data were collected from the various peptide-CaM complexes. Steady-state fluorescence intensity measurements indicated that, in the presence of an excess of calcium, both peptides bind to the calmodulin mutants with a 1:1 stoichiometry. The tryptophans located in loops I and IV exhibited red-shifted emission maxima (356 nm), high quantum yields (0.3), and long average lifetimes (6 ns). They responded in a similar manner to peptide binding, by only slight changes in their fluorescence features. In contrast, the fluorescence intensity of the tryptophans in loops II and III decreased markedly, and their fluorescence spectrum was blue-shifted upon peptide binding. Analysis of the tryptophan fluorescence decay of the last mentioned calmodulins supports a model in which the equilibrium between two (Trp-99) or three (Trp-62) states of these tryptophan residues, each characterized by a different lifetime, was altered toward the blue-shifted short lifetime component upon peptide binding. Taken together, these data provide new evidence that both lobes of calmodulin are involved in peptide binding. Both peptides induced similar changes in the fluorescence properties of the tryptophan residues located in the calcium-binding loops, with the exception of calmodulin with Trp-135. For this last mentioned calmodulin, slight differences were observed. Tryptophan in the central helix responded differently to RS20F and RS20CK binding. RS20F binding induced a red-shift in the emission maximum of Trp-81 while RS20CK induced a blue-shift. The quenching rate of Trp-81 by iodide was slightly reduced upon RS20CK binding, while RS20F induced a 2-fold increase. These results provide evidence that the environment of Trp-81 is different in each case and are, therefore, consistent with the hypothesis that the central helix can play a differential role in the recognition of, or response to, CaM-binding structures.

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

Trifluoperazine binding to mutant calmodulins.

Trifluoperazine (TFP) binding by 14 calmodulins, including 12 produced by site-directed mutagenesis, was determined. While vertebrate calmodulin binds 4.2 +/- 0.2 equiv of TFP, Escherichia coli expressed but unmutated calmodulins bind about 5.0 +/- 0.5 equiv of TFP. The cause for this difference is not known. The E. coli expressed proteins consist of two different series expressed from different calmodulin genes, CaMI and SYNCAM. The wild-type genes code for proteins that differ by nine conservative amino acid substitutions. Both these calmodulins bind 5 equiv of TFP with similar affinities, thus none of these conservative substitutions has any additional effect on TFP binding. Some altered calmodulins (deletion of EE83-84 or SEEE81-84, changing DEE118-120----KKK, M124----I,E120----K, or E82----K) have no appreciable effect on TFP binding. Other mutations affect either the binding of one TFP (deletion of E84) or about two TFP (changing E84----K, EEE82-84----KKK, E67----A, DEQ6-8----KKK, or E11----K). The mutations that affect TFP binding are localized to three regions of calmodulin: The amino-terminal alpha-helix, the central helix between the two globular ends of calmodulin, and a calcium-binding site in the second calcium-binding domain. The results are consistent with each of these regions either directly participating in drug binding or involved structurally in maintaining or inducing the correct conformation for TFP binding in the amino-terminal half of calmodulin.

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

Use of DNA sequence and mutant analyses and antisense oligodeoxynucleotides to examine the molecular basis of nonmuscle myosin light chain kinase autoinhibition, calmodulin recognition, and activity.

The first primary structure for a nonmuscle myosin light chain kinase (nmMLCK) has been determined by elucidation of the cDNA sequence encoding the protein kinase from chicken embryo fibroblasts, and insight into the molecular mechanism of calmodulin (CaM) recognition and activation has been obtained by the use of site-specific mutagenesis and suppressor mutant analysis. Treatment of chicken and mouse fibroblasts with antisense oligodeoxynucleotides based on the cDNA sequence results in an apparent decrease in MLCK levels, an altered morphology reminiscent of that seen in v-src-transformed cells, and a possible effect on cell proliferation. nmMLCK is distinct from and larger than smooth muscle MLCK (smMLCK), although their extended DNA sequence identity is suggestive of a close genetic relationship not found with skeletal muscle MLCK. The analysis of 20 mutant MLCKs indicates that the autoinhibitory and CaM recognition activities are centered in distinct but functionally coupled amino acid sequences (residues 1,068-1,080 and 1,082-1,101, respectively). Analysis of enzyme chimeras, random mutations, inverted sequences, and point mutations in the 1,082-1,101 region demonstrates its functional importance for CaM recognition but not autoinhibition. In contrast, certain mutations in the 1,068-1,080 region result in a constitutively active MLCK that still binds CaM. These results suggest that CaM/protein kinase complexes use similar structural themes to transduce calcium signals into selective biological responses, demonstrate a direct link between nmMLCK and non-muscle cell function, and provide a firm basis for genetic studies and analyses of how nmMLCK is involved in development and cell proliferation.

Amino Acid Sequence↗
PubMed1990

Analysis of the molecular basis of calmodulin defects that affect ion channel-mediated cellular responses: site-specific mutagenesis and microinjection.

The ability of microinjected calmodulin to temporarily restore an ion channel-mediated behavioral phenotype of a calmodulin mutant in Paramecium tetraurelia (cam1) is dependent on the amino acid side chain that is present at residue 101, even when there is extensive variation in the rest of the amino acid sequence. Analysis of conservation of serine-101 in calmodulin suggests that the ability of calmodulin to regulate this ion channel-associated cell function may be a biological role of calmodulin that is widely distributed phylogenetically. A series of mutant calmodulins that differ only at residue-101 were produced by in vitro site-specific mutagenesis and expression in Escherichia coli, purified to chemical homogeneity, and tested for their ability to temporarily restore a wild-type behavioral phenotype to cam1 (pantophobiacA1) Paramecium. Calmodulins with glycine-101 or tyrosine-101 had minimal activity; calmodulins with phenylalanine-101 or alanine-101 had no detectable activity. However, as a standard of comparison, all of the calmodulins were able to activate a calmodulin-regulated enzyme, myosin light chain kinase, that is sensitive to point mutations elsewhere in the calmodulin molecule. Overall, these results support the hypothesis that the structural features of calmodulin required for the transduction of calcium signals varies with the particular pathway that is being regulated and provide insight into why inherited mutations of calmodulin at residue 101 are nonlethal and selective in their phenotypic effects.

Amino Acid Sequence↗
PubMed1990

A knowledge-based experimental design system for nucleic acid engineering.

Presented in this paper is a knowledge-based experimental design system that incorporates the domain expertise used in nucleic acid engineering, thus automating the processing of error-prone, laborious low-level work, and many decision-making steps, and guiding the biologist toward a workable plan. This allows the biologist to work at a higher abstraction level, concentrating on more fundamental, difficult and challenging problems directly related to protein structure - function relationships. Cassette-based site-directed mutagenesis and synthetic gene designs are used as examples to illustrate the utility of the knowledge-based system approach to experimental design.

Algorithms↗
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↗