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T A Craig

Publications and source records attributed to T A Craig.

10 recordsLinked to original sources
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↗
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

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

Computational and site-specific mutagenesis analyses of the asymmetric charge distribution on calmodulin.

Calmodulin's calculated electrostatic potential surface is asymmetrically distributed about the molecule. Concentrations of uncompensated negative charge are localized near certain alpha-helices and calcium-binding loops. Further calculations suggest that these charge features of calmodulin can be selectively perturbed by changing clusters of phylogenetically conserved acidic amino acids in helices to lysines. When these cluster charge reversals are actually produced by using cassette-based site-specific mutagenesis of residues 82-84 or 118-120, the resulting proteins differ in their interaction with two distinct calmodulin-dependent protein kinases, myosin light chain kinase and calmodulin-dependent protein kinase II. Each calmodulin mutant can be purified to apparent chemical homogeneity by an identical purification protocol that is based on conservation of its overall properties, including calcium binding. Although cluster charge reversals result in localized perturbations of the computed negative surface, single amino acid changes would not be expected to alter significantly the distribution of the negative surface because of the relatively high density of uncompensated negative charge in the region around residues 82-84 and 118-120. However, this does not preclude the possibility of single amino acid charge perturbations having a functional effect on the more intimate, catalytically active complex. The electrostatic surface of calmodulin described in this report may be a feature that would be altered only by cluster charge reversal mutations. Overall, the results suggest that the charge properties of calmodulin are one of several properties that are important for the efficient assembly of calmodulin-protein kinase signal transduction complexes in eukaryotic cells.

Amino Acid Sequence↗
PubMed1988

A knowledge-based system for cassette mutagenesis experimental design.

A knowledge-based system for the design and planning of cassette mutagenesis experiments has been developed for scientists working in the field of structural biology and protein engineering. The system applies domain-specific knowledge to manage the menial details and automate most of the decision-making steps involved in the design process. This allows scientists to work at a high abstraction level, and results in significant time savings and increased productivity. The system also includes an automated documentation facility to improve the efficiency and accuracy of record keeping.

Artificial Intelligence↗
PubMed1987

Site-specific mutagenesis of the alpha-helices of calmodulin. Effects of altering a charge cluster in the helix that links the two halves of calmodulin.

Alteration of residues 82-84 in the alpha-helix that links the two halves of calmodulin results in a differential effect on activator activity. Previous studies (Lukas, T. J., Burgess, W. H., Prendergast, F. G., Lau, W., and Watterson, D. M. (1986) Biochemistry 25, 1458-1464) indicated the importance of positive charge clusters in the calmodulin-binding protein, myosin light chain kinase. This suggested the possible importance of complementary negative charge clusters in calmodulin. By using an efficient cassette mutagenesis approach and a synthetic calmodulin gene (Roberts, D. M., Crea, R., Malecha, M., Alvarado-Urbina, G., Chiarello, R. H., and Watterson, D. M. (1985) Biochemistry 24, 5090-5098), this possibility was directly addressed by engineering a new calmodulin, VU-8 calmodulin, in which the glutamate cluster at residues 82-84 in the synthetic gene product (VU-1 calmodulin) was replaced by three lysines. VU-8 calmodulin activated phosphodiesterase to the same maximal extent as VU-1 calmodulin, although there was an alteration in the concentration of calmodulin required for half-maximal stimulation. In contrast, myosin light chain kinase was activated to only 30% of maximal activity and NAD kinase was not activated. These results provide insight into the functional role of the unusual central helix structure found in the calmodulin family of proteins and indicate that different, although possibly overlapping, chemical complementarities are employed in the interaction between calmodulin and its various physiological targets.

Amino Acid Sequence↗
PubMed1985

Evidence for Ca++-calmodulin control of transplasmalemma electron transport in carrot cells.

Cultured carrot cells exhibit transmembrane ferricyanide reduction through a plasma membrane redox system, which may be associated with an iron reduction and uptake system in plant roots. Here we provide evidence for the inhibition of transplasma membrane ferricyanide reduction by four different Ca2+-calmodulin type antagonists, calmidazolium, trifluoperazine, pimozide and fluphenazine. These compounds inhibit in low concentrations (approximately 5-10 microM) in a time-dependent manner. Higher concentrations (50-100 microM) are required to inhibit transmembrane ferricyanide reduction in 10 min rather than in 30 min. The permeable calcium chelator, TMB-8, also inhibits transmembrane ferricyanide reduction in carrot cells. Since the redox system is controlled by hormones, the effects of anticalmodulin agents on hormone response may be mediated through the redox system.

Calcium↗
PubMed1985

Transmembrane ferricyanide reduction in carrot cells.

Carrot cells (Daucus carota) grown in tissue culture are capable of reducing the non-permeable electron acceptor, ferricyanide, with concomitant proton extrusion from the cell. Optimum conditions for transmembrane ferricyanide reduction include a pH of 7.0-7.5 in a medium containing 10 mM each KCl, NaCl and CaCl2. Data are shown to prove that transmembrane ferricyanide reduction is an enzymatic process. It does not depend on the secretion of phenolics from the cell within the time limits of the assay (10 min). The presence of broken cells and cell fragments are excluded on the basis of stimulation or only slight inhibition by mitochondrial inhibitors. However, transmembrane ferricyanide reduction by carrot cells is inhibited about 50% by various glycolysis inhibitors, which are presumed to reduce the internal levels of NADH. Treatment of cells with p-diazoniumbenzenesulfonic acid, a non-permeant membrane modifying agent, also inhibits transmembrane ferricyanide reduction more than 90%. The data presented support the existence of a transplasma membrane redox system in carrot cells.

Cell Membrane↗