scholarly journals Peptide and metal ion-dependent association of isolated helix-loop-helix calcium binding domains: Studies of thrombic fragments of calmodulin

2000 ◽  
Vol 9 (5) ◽  
pp. 964-975 ◽  
Author(s):  
Richard D. Brokx ◽  
Hans J. Vogel
Keyword(s):  
Calcium Binding ◽  
Metal Ion ◽  
Helix Loop Helix ◽  
Binding Domains

Calmodulin: a versatile calcium mediator protein

1994 ◽  
Vol 72 (9-10) ◽  
pp. 357-376 ◽  
Author(s):  
Hans J. Vogel
Keyword(s):  
Amino Acids ◽  
Intracellular Calcium ◽  
Calcium Binding ◽  
Unnatural Amino Acids ◽  
Metal Ion ◽  
Structural Information ◽  
Limited Proteolysis ◽  
Hydrophobic Surface ◽  
Site Directed Mutagenesis ◽  
Binding Domains

The level of intracellular calcium is strictly regulated in all cells. In a resting cell, the [Ca2+] is ≤ 10−7 M and during activation it rises to approximately 10−6 M. Calmodulin (CaM) is the secondary messenger protein that has to translate this modest rise in intracellular calcium into a physiological response in all eukaryotic cells. CaM can activate almost 30 different target systems, including smooth muscle contraction, protein kinases and phosphatases, nitric oxide synthases, and calcium-extruding pumps. It is an acidic protein of 148 amino acids with four helix–loop–helix calcium-binding domains and it has a characteristic dumbbell shape in the crystal structure. In this review I discuss which features of CaM allow it to be such a universal and versatile calcium regulator. First of all, the positive cooperative calcium binding to all four binding sites of CaM in the presence of a target protein allows the protein to act effectively during a calcium transient. Secondly, the high Met content of two hydrophobic surface patches on the two domains of CaM creates a flexible and pliable, yet sticky, interaction surface that does not place high demands on the specificity of the interaction. Consequently, calcium-CaM can bind effectively to the CaM-binding domains of all its target proteins, despite their lack of amino acid sequence homology; their only common feature is that they are hydrophobic basic peptides that have a propensity to form an α-helix. CaM's capacity to recognize its CaM-binding domains is further enhanced by its third crucial feature, the intrinsic flexibility of the central linker region; this allows the two domains of CaM to slide over the surface of the α-helical bound peptide, to find their most favourable binding orientation. In this review I have also presented selected examples of a variety of experimental techniques that have contributed to our understanding of this unique multitasking protein. These include studies with well-established techniques such as site-directed mutagenesis, chemical modification, limited proteolysis, circular dichroism, and two-dimensional nuclear magnetic resonance (NMR), as well as novel or less common approaches involving the use of unnatural amino acids, metal-ion NMR, lysine pKa determinations, and isotope-edited Fourier transform infrared spectroscopy. In combination with available structural information, these studies have provided considerable detail in our understanding of this versatile calcium regulatory protein.Key words: calmodulin, calcium metabolism, methionine, lysine, NMR spectroscopy.

Nuclear proteins of the bovine esophageal epithelium. I. Monoclonal antibody W2 specifically reacts with condensed nuclei of differentiated superficial cells

1993 ◽  
Vol 104 (2) ◽  
pp. 237-247 ◽  
Author(s):  
T.K. Tang ◽  
T.M. Hong ◽  
C.Y. Lin ◽  
M.L. Lai ◽  
C.H. Liu ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Metal Ion ◽  
Gradient Centrifugation ◽  
Basal Layer ◽  
Small Cells ◽  
Target Antigen ◽  
Esophageal Epithelium ◽  
Nuclear Condensation ◽  
Calcium Binding Site

Cells from three layers of the bovine esophageal epithelium, representing different stages of differentiation, were dissociated and separated by Percoll gradient centrifugation into fractions of small, medium and large sizes. A majority of the large cells possessed condensed nuclei, a characteristic feature of terminal differentiation of the superficial epithelium. The small cells resembled the proliferate cells of the basal layer. In vitro culture of the esophageal epithelial cells resulted in proliferation of the small cells, colony formation, and, in some cases, differentiation into cells with condensed nuclei. Nuclei, or nuclear subfractions derived from cells of the different layers, were used as immunogens for the generation of hybridomas secreting monoclonal antibodies that bound specifically to different regions of the esophageal tissue. One such antibody, designated W2, labeled the condensed nuclei from the superficial layer of stratified esophageal and corneal epithelia in situ, as well as the large cells from esophageal culture in vitro. Thus, the expression of the W2 antigen may be associated with the process of nuclear condensation during epithelial differentiation. Immunoisolation of the target antigen of W2 from extracts of large cells of the bovine esophagus yielded a band of M(r) approximately 33,000 on nonreducing polyacrylamide gels. This band dissociated into two polypeptides, of M(r) approximately 22,000 and approximately 11,000, upon treatment with dithiothreitol. Amino acid sequence analysis of the larger polypeptide showed extensive homology to a group of small calcium-binding proteins, including two helix-turn-helix motifs designated as the EF-hand, characteristic of the configuration of the metal-ion coordinating ligands of the calcium-binding site. Similarly, the sequence at the amino terminus of the polypeptide of approximately 11,000 indicated that it was the light chain counterpart of the same calcium-binding protein complex.

S100A13 and S100A6 exhibit distinct translocation pathways in endothelial cells

2002 ◽  
Vol 115 (15) ◽  
pp. 3149-3158 ◽  
Author(s):  
Hsiao-Ling Hsieh ◽  
Beat W. Schäfer ◽  
Jos A. Cox ◽  
Claus W. Heizmann
Keyword(s):  
Endothelial Cells ◽  
Calcium Binding ◽  
Protein Translocation ◽  
S100 Proteins ◽  
Cellular Functions ◽  
Specific Expression ◽  
Binding Domains ◽  
Cellular Environment ◽  
Ef Hands ◽  
Physiological Tasks

S100 proteins have attracted great interest in recent years because of their cell- and tissue-specific expression and association with various human pathologies. Most S100 proteins are small acidic proteins with calcium-binding domains — the EF hands. It is thought that this group of proteins carry out their cellular functions by interacting with specific target proteins, an interaction that is mainly dependent on exposure of hydrophobic patches, which result from calcium binding. S100A13, one of the most recently identified members of the S100 family, is expressed in various tissues. Interestingly,hydrophobic exposure was not observed upon calcium binding to S100A13 even though the dimeric form displays two high- and two low- affinity sites for calcium. Here, we followed the translocation of S100A13 in response to an increase in intracellular calcium levels, as protein translocation has been implicated in assembly of signaling complexes and signaling cascades, and several other S100 proteins are involved in such events. Translocation of S100A13 was observed in endothelial cells in response to angiotensin II, and the process was dependent on the classic Golgi-ER pathway. By contrast, S100A6 translocation was found to be distinct and dependent on actin-stress fibers. These experiments suggest that different S100 proteins utilize distinct translocation pathways, which might lead them to certain subcellular compartments in order to perform their physiological tasks in the same cellular environment.

HIGH AFFINITY CALCIUM BINDING TO DOMAINES OF PROTEIN C THAT ARE HOMOLOGUS TO THE EPIDERMAL GROWTH FACTOR

1987 ◽  
Author(s):  
A KÖhlin ◽  
J Stenflo
Keyword(s):  
Calcium Ions ◽  
Calcium Ion ◽  
Calcium Binding ◽  
Plasma Proteins ◽  
Metal Ion ◽  
Protein C ◽  
Protein S ◽  
Protein Z ◽  
High Affinity ◽  
Epidermal Growth

In addition to γ-carboxyglutamic acid (Gla)-dependent calcium binding all of the vitamin K-dependent plasma proteins, except prothrombin, have one or two high affinity calcium binding sites that do not require the Gla residues. A common denominator among these proteins (factors IX, X, protein C, protein Z and protein S) is that they have domaines that are homologus to the epidermal growth factor (EGF) precursor. In factors VII,IX,X, protein C and in protein Z the aminoterminal of two EGF homology regions contain one residue of β-hydroxyaspartic acid (Hya) whereas in protein S the aminoterminal EGF homology region contains Hya and the three following contain one β-hydroxyasparagine residue each.In an attempt to elucidate the role of the EGF homology regions in the Gla independent calcium binding we have isolated a tryptic fragment (residue 44-138) from the light chain of human protein C. The fragment was isolated using a monoclonal antibody that recognizes a calcium ion stabilized epitope that is expressed both in intact protein C and in protein C lacking the Gla domaine.The antibody bound the isolated EGF homology region in the presence of calcium ions but not in EDTA containing buffer. A calcium ion titration showed half maximal binding at approximately 200 μM Ca2+. The metal ion induced conformational change in the isolated fragment was also studied with affinity purified rabbit antibodies against Gla domainless protein C. Antibodies that bound in the presence of calcium ions and that could be eluted with EDTA recognized the metal ion induced conformational change in the isolated EGF homology domain. Our results suggest that one or both of the EGF homology regions are involved in the Gla-independent high affinity calcium binding in the vitamin K-dependent plasma proteins.

Locating the Metal Ion in Calcium-Binding Proteins by Using Cerium(III) as a Probe

ChemBioChem ◽  
2001 ◽  
Vol 2 (7-8) ◽  
pp. 550-558 ◽  
Author(s):  
Ivano Bertini ◽  
Yong-Min Lee ◽  
Claudio Luchinat ◽  
Mario Piccioli ◽  
Luisa Poggi
Keyword(s):  
Calcium Binding ◽  
Metal Ion ◽  
Binding Proteins ◽  
Calcium Binding Proteins

scholarly journals Putative calcium-binding domains of theCaenorhabditis elegansBK channel are dispensable for intoxication and ethanol activation

2015 ◽  
Vol 14 (6) ◽  
pp. 454-465 ◽  
Author(s):  
S. J. Davis ◽  
L. L. Scott ◽  
G. Ordemann ◽  
A. Philpo ◽  
J. Cohn ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Binding Domains
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