D. Martin Watterson

Calcium-Binding Proteins and the Molecular Basis of Calcium Action

Publisher Summary This chapter discusses the ways in which calcium is bound to a protein as well as the role of calcium in protein function. Proteins that contain γ-carboxyglutamic acid (Gla) are discussed as an example of proteins that bind calcium mainly by bidentate chelation. Prothrombin is the prototypical Gla-containing plasma protein, especially with regard to structural characteristics and calcium- and phospholipid-binding properties. Lectins are proteins that have no known enzymatic activity, but exhibit numerous biological activities that are related to their ability to bind carbohydrates in the presence of divalent cations. The proposed roles for calcium and the postulated calcium-binding sites are based on homology with concanavalin A (Con A). A variety of enzymes that catalyze the hydrolysis of ester, phosphodiester, and peptide bonds bind calcium ions. The calcium-binding hydrolytic enzymes include many hydrolytic enzymes, but mechanistic and crystallographic data are available only for phospholipase A 2 , staphylococcal nuclease, thermolysin, trypsin, and chymotrypsin. The vitamin D-dependent calcium-binding protein structure indicates that intracellular calcium-modulated proteins may utilize other structures for calcium-binding.

Structure, function, and mechanism of action of Calmodulin

The structure, function, and mechanism of action of plant calmodulins have been the subject of intense study for plant scientists during the past several years. While precedents in animal biochemistry and physiology have suggested logical starting points for studies of how calmodulin is involved in higher plant and algal cell function, recent biochemical studies have demonstrated unique structural characteristics for this highly conserved protein. Enzym‐ological analyses have demonstrated novel functional properties and provided limited insight into the molecular mechanisms of calmodulin action. This review will summarize much of the early work, but will concern itself mainly with the methods and approaches that are used to study plant and animal calmodulins as well as models for how calmodulin may be involved in plant cell function.

Characterization of human brain S100 protein fraction: amino acid sequence of S100β

Abstract: Two major components of human brain S100 fraction were purified by HPLC and an amino acid sequence was elucidated for the S100β component. Human S100 proteins showed absorption spectra and amino acid compositions similar to S100α and S100β from bovine brain. However, the relative amounts of the human proteins were 4% S100α and 96% S100β by weight, while the bovine protein distribution was 47% S100α and 53% S100β by weight. An amino acid sequence of human S100β was established by analysis of overlapping fragments generated by cyanogen bromide and trypsin cleavage. Three amino acid sequence differences between the human and bovine S100β were found at residues 7, 62, and 80. These differences were chemically conservative and compatible with minimum single base changes in the codon structures. These results document that S100β is a conserved protein among mammals and provide the necessary foundation for current clinical studies.

Analysis of differences between Coomassie blue stain and silver stain procedures in polyacrylamide gels: Conditions for the detection of calmodulin and troponin C

It is reported that the conditions used in some silver stain procedures can fail to detect calmodulin, troponin C, and other proteins with similar physical properties. Conditions are described that allow the reproducible detection of these proteins. Two phenomena are described: (1) lack of protein staining when treatment with glutaraldehyde is omitted from the protocol, and (2) loss of small proteins from the gel matrix during prolonged washing procedures. These data directly demonstrate that the use of some silver staining protocols can result in misleading data in biological studies and provide an explanation for at least one class of proteins of how silver staining and Coomassie blue staining of gels can give different results.

Isolation and characterization of calmodulin from the motile green alga Chlamydomonas reinhardtii

Calmodulin, a calcium-binding protein with no known enzymatic activity but multiple, in vitro effector activities, has been purified to apparent homogeneity from the unicellular green alga Chlamydomonas reinhardtii and compared to calmodulin from vertebrates and higher plants. Chlamydomonas calmodulin was characterized in terms of electrophoretic mobility, amino acid composition, limited amino acid sequence analysis, immunoreactivity, and phosphodiesterase activation. Chlamydomonas calmodulin has two histidine residues similar to calmodulin from the protozoan Tetrahymena. However, unlike the protozoan calmodulin, only one of the histidinyl residues of Chlamydomonas calmodulin is found in the COOH-terminal third of the molecule. Chlamydomonas calmodulin lacks trimethyllysine but does have a lysine residue at the amino acid sequence position corresponding to the trimethyllysine residue in bovine brain and spinach calmodulins. The lack of this post-translational modification does not prevent Chlamydomonas calmodulin from quantitatively activating bovine brain phosphodiesterase. These studies also demonstrate that this unique calmodulin from a phylogenetically earlier eukaryote may be as similar to vertebrate calmodulin as it is to higher plant calmodulins, and suggest that Chlamydomonas calmodulin may more closely approximate the characteristics of a putative precursor of the calmodulin family than any calmodulin characterized to date.

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.

AN INTERDISCIPLINARY APPROACH TO THE MOLECULAR MECHANISMS OF CALMODULIN ACTION: COMPARATIVE BIOCHEMISTRY, SITE-SPECIFIC MUTAGENESIS, AND PROTEIN ENGINEERING

Publisher Summary This chapter presents an interdisciplinary approach to the molecular mechanisms of calmodulin action. The VU-1 calmodulin has an amino acid sequence that is a hybrid of vertebrate and plant calmodulins. The bacterially expressed protein lacks two of the post-translational modifications common to vertebrate and some other calmodulins. These include the acetylation of the amino terminus and the trimethylation of lysine-115. Apart from the lack of acetylation, the changes in the VU-1 calmodulin structure have been observed in the naturally occurring calmodulins. The study of calmodulin-activated proceses by protein engineering requires structural information about specific target enzymes. The detailed studies have focused on the calmodulin dependent enzyme Myosin Light Chain Kinase (MLCK). The amino acid sequences of potential calmodulin binding sites have been elucidated for skeletal and smooth muscle MLCK. A new calmodulin expression vector that allows for mutagenesis, amplification, characterization and expression of mutant calmodulin genes in E. coli has been constructed. The new expression vector, pVUCH-1, is a hybrid of pVUC-1 and pUC8. The orientation of the gene relative to the tac promoter elements within the plasmid is the same as that found in pVUC-1.

Structural characterization of the calcium binding protein S100 from adipose tissue

A partial amino acid sequence for bovine adipose tissue S100 was elucidated by characterization of peptides generated by cyanogen bromide cleavage. The cyanogen bromide peptides were aligned by homology with the bovine brain S100 beta sequence. The results demonstrate that adipose S100 beta is probably identical to brain S100 beta, and suggest that S100 beta is a conserved protein among tissues of the same species.

Molecular Mechanisms of Calmodulin Action

Calcium has many biological functions and several groups of macromolecules have the ability to bind calcium with various selectivities and affinities. A rapidly expanding body of knowledge about the structure, thermodynamic and kinetic properties, and cell biology of calcium binding proteins strongly indicates that the targets or receptors for calcium acting as a signal transducer in eukaryotic cells are a class of calcium binding proteins referred to as calcium modulated proteins. These proteins are characterized by their ability to bind calcium in a reversible manner with dissociation constants in the nanomolar to micromolar range under physiological conditions. Although it is not possible yet to predict with any degree of certainty what type of calcium binding structure or molecular mechanism might be associated with a given biological function of calcium, a trend has begun to emerge from the detailed analyses of calcium modulated proteins1

Amino acid sequence of the phosphorylation site of bovine cardiac myosin light chain

Amino acid sequences of peptides containing the phosphorylation site of bovine cardiac myosin light chain (L2) were determined. The site was localized to a serine residue in the tentative amino terminus of the light chain and is homologous to phosphorylation sites in other myosin light chains. Phosphorylation of bovine cardiac light chain by chicken gizzard myosin light chain kinase was Ca2+-calmodulin dependent. Kinetic data gave a Km of 107; microM and a Vmax of 23.6 mumol min-1 mg-1. In contrast to what has been observed with smooth muscle light chains, neither the phosphorylation site fragment of the cardiac light chain nor a synthetic tetradecapeptide containing the phosphorylation site were effectively phosphorylated by the chicken gizzard kinase. Phosphorylation of cardiac myosin light chains by chicken gizzard myosin light chain kinase, therefore, requires other regions of the light chain in addition to a phosphate acceptor site.