scholarly journals EF-hand motifs of α, β and γ isoforms of diacylglycerol kinase bind calcium with different affinities and conformational changes

1997 ◽  
Vol 321 (1) ◽  
pp. 59-64 ◽  
Author(s):  
Keiko YAMADA ◽  
Fumio SAKANE ◽  
Norio MATSUSHIMA ◽  
Hideo KANOH
Keyword(s):  
Conformational Changes ◽  
Calcium Binding ◽  
Equilibrium Dialysis ◽  
Diacylglycerol Kinase ◽  
Intrinsic Properties ◽  
Apparent Dissociation Constant ◽  
Ef Hand ◽  
Order Of Magnitude ◽  
Hydrophobic Amino Acids ◽  
Ef Hands

The three diacylglycerol kinase isoenzymes (DGKα, DGKβ and DGKγ) cloned so far contain in common a tandem repeat of EF-hand motifs. However, the Ca2+ dependences of the DGK activities are known to be variable between isoenzymes, and the Ca2+-binding activities of these motifs have not been tested except for those present in DGKα. We therefore attempted to define the intrinsic properties of EF-hands occurring in the DGK isoenzymes. For this purpose we bacterially expressed and purified the EF-hand motifs (termed DKE forms) of the three DGKs. Equilibrium dialysis with the purified DKE forms showed that all of the expressed proteins could bind approx. 2 mol of Ca2+ per mol. However, the apparent dissociation constant (Kd) for calcium binding to α-DKE (9.9 µM) was an order of magnitude greater than those estimated for β-DKE (0.89 µM) and γ-DKE (0.40 µM). Experiments with 2-p-toluidinylnaphthalene 6-sulphonate, a probe for hydrophobic regions of proteins, showed that the binding of Ca2+ to β-DKE resulted in the exposure of hydrophobic amino acids, whereas hydrophobic regions of α-DKE and γ-DKE were masked by the addition of Ca2+. Taken together, these results indicate that DGKα, DGKβ and DGKγ possess EF-hand structures with intrinsic properties different from each other with respect to affinities for Ca2+ and Ca2+-induced conformational changes.

scholarly journals The binding of calcium to a salivary phosphoprotein, protein C, and comparison with calcium binding to protein A, a related salivary phosphoprotein

1977 ◽  
Vol 163 (2) ◽  
pp. 241-245 ◽  
Author(s):  
A Bennick
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Alkaline Phosphatase ◽  
Magnetic Resonance ◽  
Conformational Changes ◽  
Calcium Binding ◽  
Protein C ◽  
Equilibrium Dialysis ◽  
Protein A ◽  
Apparent Dissociation Constant ◽  
Protein Molecules

The binding of Ca2+ to a salivary phosphoprotein, protein C, was studied by equilibrium dialysis. In 5mM-Tris/HCl buffer, pH 7.5, protein C bound 190 nmol of Ca2+/mg of protein. The apparent dissociation constant, K, was determined to be 1.9 x 10(-4)M and the binding of Ca2+ to the protein was non-co-operative. The binding of Ca2+ to protein C apparently depends on groups which ionize above pH 5.0. Ca2+ binding decreased with increased concentration of the dialysis buffer and on addition of SrCL2, MgCl2 and MnCl2 to the dialysis buffer. Digestion of protein C with trypsin or collagenase or heating of the protein to 60 degrees or 100 degrees C had little or no effect on the Ca2+ binding. Digestion of protein C with alkaline phosphatase caused a decrease in the amount of protein-bound Ca2+. This was also found for another salivary phosphoprotein, protein A. In the absence of Ca2+ the S020,w for protein C was 1.29 S and in the presence of Ca2+ it was 1.46S. Ca2+ may cause a conformational change in the protein or an aggregation of the protein molecules. No conformational changes of protein C in the presence of Ca2+ could be detected by circular dichroism or nuclear magnetic resonance.

scholarly journals Calmodulin and Calmodulin Binding Proteins in Dictyostelium: A Primer

2020 ◽  
Vol 21 (4) ◽  
pp. 1210
Author(s):  
Danton H. O’Day ◽  
Ryan J. Taylor ◽  
Michael A. Myre
Keyword(s):  
Conformational Changes ◽  
Calcium Binding ◽  
Binding Proteins ◽  
Regulatory Protein ◽  
Model Organism ◽  
Ion Binding ◽  
Calcium Dependent ◽  
Calmodulin Binding ◽  
Human Counterpart ◽  
Ef Hands

Dictyostelium discoideum is gaining increasing attention as a model organism for the study of calcium binding and calmodulin function in basic biological events as well as human diseases. After a short overview of calcium-binding proteins, the structure of Dictyostelium calmodulin and the conformational changes effected by calcium ion binding to its four EF hands are compared to its human counterpart, emphasizing the highly conserved nature of this central regulatory protein. The calcium-dependent and -independent motifs involved in calmodulin binding to target proteins are discussed with examples of the diversity of calmodulin binding proteins that have been studied in this amoebozoan. The methods used to identify and characterize calmodulin binding proteins is covered followed by the ways Dictyostelium is currently being used as a system to study several neurodegenerative diseases and how it could serve as a model for studying calmodulinopathies such as those associated with specific types of heart arrythmia. Because of its rapid developmental cycles, its genetic tractability, and a richly endowed stock center, Dictyostelium is in a position to become a leader in the field of calmodulin research.

Calcium ion binding to clinically relevant chemical modifications of human serum albumin

1995 ◽  
Vol 41 (11) ◽  
pp. 1654-1661 ◽  
Author(s):  
H Vorum ◽  
K Fisker ◽  
M Otagiri ◽  
A O Pedersen ◽  
U Kragh-Hansen
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Conformational Changes ◽  
Calcium Binding ◽  
Equilibrium Dialysis ◽  
Thiol Group ◽  
Protein Molecule ◽  
Penicillin G ◽  
Lysine Residues

Abstract Calcium binding to glycated, penicilloylated, acetylated, and normal defatted human serum albumin as well as to mercapt- and nonmercaptalbumin was studied by equilibrium dialysis of radioactive Ca2+. Binding was quantified by five Scatchard constants [ni = 1, (i = 1-4) and n5 = 10]. Glycation resulted in increased k1- and k2-values and unchanged k3-k5-values, whereas penicilloylation increased all five association constants. The increments were greater the more pronounced the modification, and the enhancements caused by penicilloylation were, for the same degree of modification, greater than those produced by glycation. In contrast, acetylation by acetylsalicylate did not affect calcium binding. Likewise, binding to mercapt- and nonmercaptalbumin was the same, a finding showing that the thiol group of cysteine 34 is not important for calcium binding. D-Glucose and penicillin G are known to react with lysine residues of albumin, and the enhancement of binding resulting from glycation or penicilloylation is probably brought about by unspecific electrostatic effects, possibly supplemented by conformational changes of the protein molecule. The relative importance of the three domains of human serum albumin for calcium binding is discussed.

scholarly journals Crystal structure and calcium‐induced conformational changes of diacylglycerol kinase α EF‐hand domains

2019 ◽  
Vol 28 (4) ◽  
pp. 694-706 ◽  
Author(s):  
Daisuke Takahashi ◽  
Kano Suzuki ◽  
Taiichi Sakamoto ◽  
Takeo Iwamoto ◽  
Takeshi Murata ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Diacylglycerol Kinase ◽  
Ef Hand

scholarly journals Mutational analysis of centrin: an EF-hand protein associated with three distinct contractile fibers in the basal body apparatus of Chlamydomonas.

1992 ◽  
Vol 119 (6) ◽  
pp. 1613-1624 ◽  
Author(s):  
B E Taillon ◽  
S A Adler ◽  
J P Suhan ◽  
J W Jarvik
Keyword(s):  
Amino Acid ◽  
Basal Body ◽  
Calcium Binding ◽  
Mutational Analysis ◽  
Structural Defects ◽  
Wild Type ◽  
Immunofluorescence Analysis ◽  
Ef Hand ◽  
Level 1 ◽  
Ef Hands

Centrin, a 20-kD phosphoprotein with four calcium-binding EF-hands, is present in the centrosome/basal body apparatus of the green alga Chlamydomonas reinhardtii in three distinct locations: the nucleus-basal body connectors, the distal striated fibers, and the flagellar transition regions. In each location, centrin is found in fibrous structures that display calcium-mediated contraction. The mutant vfl2 has structural defects at all of these locations and is defective for basal body localization and/or segregation. We show that the vfl2 mutation is a G-to-A transition in the centrin structural gene which converts a glutamic acid to a lysine at position 101, the first amino acid of the E-helix of the protein's third EF-hand. This proves that centrin is required to construct the nucleus-basal body connectors, the distal striated fibers, and the flagellar transition regions, and it demonstrates the importance of amino acid 101 to normal centrin function. Based on immunofluorescence analysis using anti-centrin antibodies, it appears that vfl2 centrin is capable of binding to the basal body but is incapable of polymerizing into filamentous structures. 19 phenotypic revertants of vfl2 were isolated, and 10 of them, each of which had undergone further mutation at codon 101, were examined in detail. At the DNA level, 1 of the 10 was wild type, and the other 9 were pseudorevertants encoding centrins with the amino acids asparagine, threonine, methionine, or isoleucine at position 101. No ultrastructure defects were apparent in the revertants with asparagine or threonine at position 101, but in those with methionine or isoleucine at position 101, the distal striated fibers were found to be incomplete, indicating that different amino acid substitutions at position 101 can differentially affect the assembly of the three distinct centrin-containing fibrous structures associated with the Chlamydomonas centrosome.

scholarly journals Molecular Players of EF-hand Containing Calcium Signaling Event in Plants

2019 ◽  
Vol 20 (6) ◽  
pp. 1476 ◽  
Author(s):  
Tapan Mohanta ◽  
Dhananjay Yadav ◽  
Abdul Khan ◽  
Abeer Hashem ◽  
Elsayed Abd_Allah ◽  
...  
Keyword(s):  
Calcium Signaling ◽  
Protein Kinases ◽  
Calcium Binding ◽  
Life Forms ◽  
Terrestrial Environment ◽  
Calcium Dependent ◽  
Ef Hand ◽  
Dependent Protein ◽  
Calcium Dependent Protein Kinases ◽  
Ef Hands

Ca2+ is a universal second messenger that plays a pivotal role in diverse signaling mechanisms in almost all life forms. Since the evolution of life from an aquatic to a terrestrial environment, Ca2+ signaling systems have expanded and diversified enormously. Although there are several Ca2+ sensing molecules found in a cell, EF-hand containing proteins play a principal role in calcium signaling event in plants. The major EF-hand containing proteins are calmodulins (CaMs), calmodulin like proteins (CMLs), calcineurin B-like (CBL) and calcium dependent protein kinases (CDPKs/CPKs). CaMs and CPKs contain calcium binding conserved D-x-D motifs in their EF-hands (one motif in each EF-hand) whereas CMLs contain a D-x3-D motif in the first and second EF-hands that bind the calcium ion. Calcium signaling proteins form a complex interactome network with their target proteins. The CMLs are the most primitive calcium binding proteins. During the course of evolution, CMLs are evolved into CaMs and subsequently the CaMs appear to have merged with protein kinase molecules to give rise to calcium dependent protein kinases with distinct and multiple new functions. Ca2+ signaling molecules have evolved in a lineage specific manner with several of the calcium signaling genes being lost in the monocot lineage.

scholarly journals X-ray, spectroscopic and normal-mode dynamics of calexcitin: structure–function studies of a neuronal calcium-signalling protein

2015 ◽  
Vol 71 (3) ◽  
pp. 615-631 ◽  
Author(s):  
P. T. Erskine ◽  
A. Fokas ◽  
C. Muriithi ◽  
H. Rehman ◽  
L. A. Yates ◽  
...  
Keyword(s):  
Potassium Channel ◽  
Normal Mode ◽  
Calcium Ions ◽  
Calcium Binding ◽  
Calcium Binding Site ◽  
X Ray ◽  
Terminal Domain ◽  
Ef Hand ◽  
Ef Hands ◽  
Photoreceptor Neurons

The protein calexcitin was originally identified in molluscan photoreceptor neurons as a 20 kDa molecule which was up-regulated and phosphorylated following a Pavlovian conditioning protocol. Subsequent studies showed that calexcitin regulates the voltage-dependent potassium channel and the calcium-dependent potassium channel as well as causing the release of calcium ions from the endoplasmic reticulum (ER) by binding to the ryanodine receptor. A crystal structure of calexcitin from the squidLoligo pealeishowed that the fold is similar to that of another signalling protein, calmodulin, the N- and C-terminal domains of which are known to separate upon calcium binding, allowing interactions with the target protein. Phosphorylation of calexcitin causes it to translocate to the cell membrane, where its effects on membrane excitability are exerted and, accordingly,L. pealeicalexcitin contains two protein kinase C phosphorylation sites (Thr61 and Thr188). Thr-to-Asp mutations which mimic phosphorylation of the protein were introduced and crystal structures of the corresponding single and double mutants were determined, which suggest that the C-terminal phosphorylation site (Thr188) exerts the greatest effects on the protein structure. Extensive NMR studies were also conducted, which demonstrate that the wild-type protein predominantly adopts a more open conformation in solution than the crystallographic studies have indicated and, accordingly, normal-mode dynamic simulations suggest that it has considerably greater capacity for flexible motion than the X-ray studies had suggested. Like calmodulin, calexcitin consists of four EF-hand motifs, although only the first three EF-hands of calexcitin are involved in binding calcium ions; the C-terminal EF-hand lacks the appropriate amino acids. Hence, calexcitin possesses two functional EF-hands in close proximity in its N-terminal domain and one functional calcium site in its C-terminal domain. There is evidence that the protein has two markedly different affinities for calcium ions, the weaker of which is most likely to be associated with binding of calcium ions to the protein during neuronal excitation. In the current study, site-directed mutagenesis has been used to abolish each of the three calcium-binding sites of calexcitin, and these experiments suggest that it is the single calcium-binding site in the C-terminal domain of the protein which is likely to have a sensory role in the neuron.

Conformational changes and calcium binding by calretinin and its recombinant fragments containing different sets of EF hand motifs

Biochemistry ◽  
1995 ◽  
Vol 34 (47) ◽  
pp. 15389-15394 ◽  
Author(s):  
Jacek Kuznicki ◽  
Kenneth I. Strauss ◽  
David M. Jacobowitz
Keyword(s):  
Conformational Changes ◽  
Calcium Binding ◽  
Ef Hand ◽  
Recombinant Fragments
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