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

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 EF-Hand Ca2+-binding Protein p22 Plays a Role in Microtubule and Endoplasmic Reticulum Organization and Dynamics with Distinct Ca2+-binding Requirements

2004 ◽  
Vol 15 (2) ◽  
pp. 481-496 ◽  
Author(s):  
Josefa Andrade ◽  
Hu Zhao ◽  
Brian Titus ◽  
Sandra Timm Pearce ◽  
Margarida Barroso
Keyword(s):  
Endoplasmic Reticulum ◽  
Conformational Changes ◽  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Network Formation ◽  
Binding Protein ◽  
Dependent Manner ◽  
Microtubule Depolymerization ◽  
Independent Manner ◽  
Ef Hand

We have reported that p22, an N-myristoylated EF-hand Ca2+-binding protein, associates with microtubules and plays a role in membrane trafficking. Here, we show that p22 also associates with membranes of the early secretory pathway membranes, in particular endoplasmic reticulum (ER). On binding of Ca2+, p22's ability to associate with membranes increases in an N-myristoylation-dependent manner, which is suggestive of a nonclassical Ca2+-myristoyl switch mechanism. To address the intracellular functions of p22, a digitonin-based “bulk microinjection” assay was developed to load cells with anti-p22, wild-type, or mutant p22 proteins. Antibodies against a p22 peptide induce microtubule depolymerization and ER fragmentation; this antibody-mediated effect is overcome by preincubation with the respective p22 peptide. In contrast, N-myristoylated p22 induces the formation of microtubule bundles, the accumulation of ER structures along the bundles as well as an increase in ER network formation. An N-myristoylated Ca2+-binding p22 mutant, which is unable to undergo Ca2+-mediated conformational changes, induces microtubule bundling and accumulation of ER structures along the bundles but does not increase ER network formation. Together, these data strongly suggest that p22 modulates the organization and dynamics of microtubule cytoskeleton in a Ca2+-independent manner and affects ER network assembly in a Ca2+-dependent manner.

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