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1.
    
We have classified 865 sequences of EF‐hand proteins from five proteomes into 156 subfamilies. These subfamilies were put into six groups. Evolutionary relationships among subfamilies and groups were analyzed from the inferred ancestral sequence for each subfamily. CTER, CPV, and PEF groups arose from a common EF‐lobe (pair of adjacent EF‐hands). They have two or more EF‐lobes; the relative positions of their EF‐lobes differ from each other. Comparisons of the ancestral sequences and the inferred structures of the EF‐lobes of these groups indicate that the mutual positions of EF‐lobes were established soon after divergence of an EF‐lobe for each group and before the duplication and fusion of EF‐lobe gene(s). These ancestral sequences reveal that some subfamilies in low similarity and isolated groups did not evolve from the EF‐lobe precursor, even if their conformations are similar to the canonical EF‐hand. This is an example of convergent evolution.  相似文献   

2.
    
ALG‐2 (apoptosis‐linked gene 2) is an apoptosis‐linked calcium‐binding protein with five EF‐hand motifs in the C‐terminal region. N‐terminally truncated ALG‐2 (des3‐23ALG‐2) was crystallized by the vapour‐diffusion method in buffer consisting of either 50 mM MES pH 6.5, 12.5%(v/v) 2‐propanol and 150 mM calcium acetate or 100 mM MES pH 6.0, 15%(v/v) ethanol and 200 mM zinc acetate. Crystals of the Ca2+‐bound form belonged to space group P212121, with unit‐cell parameters a = 54.8, b = 154.4, c = 237.7 Å, α = β = γ = 90°, and diffracted to 3.1 Å resolution. Crystals of the Zn2+‐bound form belonged to space group P212121, with unit‐cell parameters a = 52.8, b = 147.5, c = 230.7 Å, α = β = γ = 90°, and diffracted to 3.3 Å resolution. The structures of the Ca2+‐bound form and the Zn2+‐bound form were solved by the molecular‐replacement method. Although both crystals contained eight ALG‐2 molecules per asymmetric unit, the metal‐ion locations and octameric arrangements were found to be significantly different.  相似文献   

3.
    
Fragment complementation has been used to investigate the role of chain connectivity in the catalytic reaction of phosphomannomutase/phosphoglucomutase (PMM/PGM) from Pseudomonas aeruginosa, a human pathogen. A heterodimer of PMM/PGM, created from fragments corresponding to its first three and fourth domains, was constructed and enzyme activity reconstituted. NMR spectra demonstrate that the fragment corresponding to the fourth (C‐terminal) domain exists as a highly structured, independent folding domain, consistent with its varied conformation observed in enzyme–substrate complexes. Steady‐state kinetics and thermodynamics studies reported here show that complete conformational freedom of Domain 4, because of the break in the polypeptide chain, is deleterious to catalytic efficiency primarily as a consequence of increased entropy. This extends observations from studies of the intact enzyme, which showed that the degree of flexibility of a hinge region is controlled by the precise sequence of amino acids optimized through evolutionary constraints. This work also sheds light on the functional advantage gained by combining separate folding domains into a single polypeptide chain.  相似文献   

4.
报告蛋白片段互补及功能重建技术是对传统的酵母双杂交技术的改进。其原理是将报告蛋白分割成两个没有功能的片段,分别与两个待检测的蛋白质融合,如果待检测的两个蛋白质能够发生相互作用,就可以使报告蛋白的两个片段发生互补,从而使其功能得以重建。这一技术在检测方法和适用的细胞类型上都对酵母双杂交系统进行了扩展。  相似文献   

5.
    
In addition to the well‐known Ca2+ sensor calmodulin, plants possess many calmodulin‐like proteins (CMLs) that are predicted to have specific roles in the cell. Herein, we described the biochemical and biophysical characterization of recombinant Arabidopsis thaliana CML14. We applied isothermal titration calorimetry to analyze the energetics of Ca2+ and Mg2+ binding to CML14, and nuclear magnetic resonance spectroscopy, together with intrinsic and ANS‐based fluorescence, to evaluate the structural effects of metal binding and metal‐induced conformational changes. Furthermore, differential scanning calorimetry and limited proteolysis were used to characterize protein thermal and local stability. Our data demonstrate that CML14 binds one Ca2+ ion with micromolar affinity (Kd ～ 12 µM) and the presence of 10 mM Mg2+ decreases the Ca2+ affinity by ～5‐fold. Although binding of Ca2+ to CML14 increases protein stability, it does not result in a more hydrophobic protein surface and does not induce the large conformational rearrangement typical of Ca2+ sensors, but causes only localized structural changes in the unique functional EF‐hand. Our data, together with a molecular modelling prediction, provide interesting insights into the biochemical properties of Arabidopsis CML14 and may be useful to direct additional studies aimed at understanding its physiological role.  相似文献   

6.
    
Chlamydomonas reinhardtii centrin is a member of the EF‐hand calcium‐binding superfamily. It is found in the basal body complex and is important for flagellar motility. Like other members of the EF‐hand family, centrin interacts with and modulates the function of other proteins in a calcium‐dependent manner. To understand how C. reinhardtii centrin interacts with its protein targets, it has been crystallized in the presence of the model peptide melittin and X‐ray diffraction data have been collected to 2.2 Å resolution. The crystals are orthorhombic, with unit‐cell parameters a = 52.1, b = 114.4, c = 34.8 Å, and are likely to belong to space group P21212.  相似文献   

7.
    
Calcineurin B homologous protein 1 (CHP1), also known as p22, is a calcium‐binding protein that plays a role in membrane trafficking and binds to multiple effector proteins, including Na+/H+ exchangers, serine/threonine protein kinase and calcineurin, potentially modulating their function. CHP1 has been crystallized at 277 K using polyethylene glycol as a precipitant. The crystal belongs to space group P21, with unit‐cell parameters a = 55.5, b = 38.5, c = 90.0 Å, β = 90.7°. A full set of diffraction data was collected to 2.2 Å resolution at 100 K using the Photon Factory synchrotron‐radiation source.  相似文献   

8.
    
Many essential physiological processes are regulated by the modulation of calcium concentration in the cell. The EF‐hand proteins represent a superfamily of calcium‐binding proteins involved in calcium signaling and homeostasis. Secretagogin is a hexa‐EF‐hand protein that is highly expressed in pancreatic islet of Langerhans and neuroendocrine cells and may play a role in the trafficking of secretory granules. We present the X‐ray structure of Danio rerio secretagogin, which is 73% identical to human secretagogin, in calcium‐free form at 2.1‐Å resolution. Secretagogin consists of the three globular domains each of which contains a pair of EF‐hand motifs. The domains are arranged into a V‐shaped molecule with a distinct groove formed at the interface of the domains. Comparison of the secretagogin structure with the solution structure of calcium‐loaded calbindin D28K revealed a striking difference in the spatial arrangement of their domains, which involves ～180° rotation of the first globular domain with respect to the module formed by the remaining domains. Proteins 2009. © 2009 Wiley‐Liss, Inc.  相似文献   

9.
    
A new family of calcineurin B‐like calcium‐binding proteins has recently been identified in Arabidopsis thaliana. AtCBL2, a member of this family, has been crystallized in the presence of calcium ions using polyethylene glycol as a precipitant at 293 K. The crystals belong to space group C2221, with unit‐cell parameters a = 83.9, b = 118.1, c = 49.1 Å. The asymmetric unit contains one molecule, with a VM of 2.36 Å3 Da−1 and a solvent content of 48%. Native diffraction data to 2.1 Å resolution have been collected using synchrotron radiation at SPring‐8.  相似文献   

10.
    
Recombinant human grancalcin, a calcium‐binding protein from leukocytes, has been crystallized in the presence or absence of Ca2+ by the vapor‐diffusion method. Two crystal forms of apo grancalcin were obtained: space group P21, with unit‐cell parameters a = 48.4, b = 81.1, c = 46.6 Å, β = 111.3°, diffracting to 1.9 Å, and space group C2, with unit‐cell parameters a = 97.0, b = 51.9, c = 75.9 Å, β = 108.5°, diffracting to 2.4 Å. Crystals were also grown in the presence of 5 mM Ca2+. They also belong to space group C2, with unit‐cell parameters a = 97.4, b = 50.3, c = 77.6 Å, β = 108.2°, which are very similar to the second apo grancalcin form. These crystals diffract to 2.5 Å.  相似文献   

11.
We have developed a method to place an EF‐lobe in a coordinate system that recognizes the similarity of its two EF‐hand domains as well as their relationship by a pseudo‐two fold axis, z. The x‐axis connects the center of mass, calculated from α‐carbons of helices E1 and F1, with the center of mass of E2 and F2. The resulting coordinate system is intrinsic to each EF‐lobe and requires no comparison with other EF‐lobes. It has provided an intuitive and informative way to compare EF‐lobes and especially those changes associated with calcium and/or target binding. We analyzed the EF‐lobes of calmodulin and of other subfamilies with four EF‐hands. We have rationalized a complex pattern of changes of conformation associated with calcium coordination and effector binding as observed in different subfamilies of EF‐hand proteins. Proteins 2012. © 2012 Wiley Periodicals, Inc.  相似文献   

12.
The relative significance of weak non-covalent interactions in biological context has been much debated. Here, we have addressed the contribution of Coulombic interactions to protein stability and assembly experimentally. The sweet protein monellin, a non-covalently linked heterodimeric protein, was chosen for this study because of its ability to spontaneously reconstitute from separated fragments. The reconstitution of monellin mutants containing large surface charge perturbations was compared to the thermostability of structurally equivalent single-chain monellin containing the same sets of mutations under varying salt concentrations. The affinity between monellin fragments is found to correlate with the thermostability of single chain monellin, indicating the involvement of the same underlying Coulombic interactions. This confirms that there are no principal differences in the interactions involved in folding and binding. Based on comparison with a previous mutational study involving hydrophobic core residues, the relative contribution of Coulombic interactions to stability and affinity is modest. However, the Coulombic perturbations only affect the association rates of reconstitution in contrast to perturbations involving hydrophobic residues, which affect primarily the dissociation rates. These results indicate that Coulombic interactions are likely to be of main importance for the association of protein assembly, relevant for functions of proteins.  相似文献   

13.
    
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14.
Microtubules (MTs) are integral to numerous cellular functions, such as cell adhesion, differentiation and intracellular transport. Their dynamics are largely controlled by diverse MT‐interacting proteins, but the signalling mechanisms that regulate these interactions remain elusive. In this report, we identify a rapid, calcium‐regulated switch between MT plus end interaction and lattice binding within the carboxyl terminus of BPAG1n4. This switch is EF‐hand dependent, and mutations of the EF‐hands abolish this dynamic behaviour. Our study thus uncovers a new, calcium‐dependent regulatory mechanism for a spectraplakin, BPAG1n4, at the MT plus end.  相似文献   

15.
    
Calexcitin was first identified in the marine snail Hermissenda crassicornis as a neuronal‐specific protein that becomes upregulated and phosphorylated in associative learning. Calexcitin possesses four EF‐hand motifs, but only the first three (EF‐1 to EF‐3) are involved in binding metal ions. Past work has indicated that under physiological conditions EF‐1 and EF‐2 bind Mg2+ and Ca2+, while EF‐3 is likely to bind only Ca2+. The fourth EF‐hand is nonfunctional owing to a lack of key metal‐binding residues. The aim of this study was to use a crystallographic approach to determine which of the three metal‐binding sites of calexcitin is most readily replaced by exogenous metal ions, potentially shedding light on which of the EF‐hands play a `sensory' role in neuronal calcium signalling. By co‐crystallizing recombinant calexcitin with equimolar Gd3+ in the presence of trace Ca2+, EF‐1 was shown to become fully occupied by Gd3+ ions, while the other two sites remain fully occupied by Ca2+. The structure of the Gd3+–calexcitin complex has been refined to an R factor of 21.5% and an Rfree of 30.4% at 2.2 Å resolution. These findings suggest that EF‐1 of calexcitin is the Ca2+‐binding site with the lowest selectivity for Ca2+, and the implications of this finding for calcium sensing in neuronal signalling pathways are discussed.  相似文献   

16.
    
Calmodulin (CaM), the ubiquitous, eukaryotic, bilobal calcium-binding regulatory protein, has been cleaved by thrombin to create two fragments. TM1 (1-106) and TM2 (107-148). NMR and CD results indicate that TMI and TM2 can associate in the presence of Ca2+ to form a complex similar to native CaM, even though the cleavage site is not in the linker region between two helix-loop-helix domains, but rather within an alpha-helix. Cadmium-113 NMR results show that this complex has enhanced metal-ion binding properties when compared to either TM1 or TM2 alone. This complex can bind several CaM-binding target peptides, as shown by gel bandshift assays, circular dichroism spectra, and 13C NMR spectra of biosynthetically methyl-13C-Met-labeled TM1 and TM2; moreover, gel bandshift assays show that the addition of a target peptide strengthens the interactions between TM1 and TM2 and increases the stability of the complex. Cadmium-113 NMR spectra indicate that the TM1:TM2 complex can also bind the antipsychotic drug trifluoperazine. However, in contrast to CaM:peptide complexes, the TM1:TM2:peptide complexes are disrupted by 4 M urea; moreover, TM1 and TM2 in combination are unable to activate CaM-dependent enzymes. This suggests that TM1:TM2 mixtures cannot bind target molecules as tightly as intact CaM, or perhaps that binding occurs but additional interactions with the target enzymes that are necessary for proper activation are perturbed by the proteolytic cleavage. The results presented here reflect the importance of the existence of helix-loop-helix Ca2+-binding domains in pairs in proteins such as CaM, and extend the understanding of the association of such domains in this class of proteins in general.  相似文献   

17.
    
Sorcin is a 198 amino‐acid Ca2+‐binding protein that belongs to the penta‐EF‐hand family. Its Ca2+‐binding domain (residues 33–198) has been crystallized in the absence of Ca2+ in two different crystal forms. Two complete data sets have been collected on a synchrotron source under cryocooling conditions from crystals grown using ammonium sulfate as precipitant: monoclinic crystals in space group C2, with unit‐cell parameters a = 130.93, b = 103.85, c = 78.55 Å, β = 118.0°, diffracting to 2.1 Å, and tetragonal crystals in space group P4212, with unit‐cell parameters a = b = 103.33, c = 79.15, diffracting to 2.7 Å. Crystals were also grown using PEG 6000 as precipitating agent. They also belong to space group C2, diffract to 2.8 Å and their unit‐cell parameters are very similar to the first form. Structure determination by molecular replacement has been initiated. Structural information should be useful for elucidating the interaction of sorcin with membrane targets.  相似文献   

18.
    
Centrins belong to a family of Ca2+‐binding EF‐hand proteins that play a fundamental role in centrosome duplication and the function of cilia. To shed light on the structure–function relationship of these proteins, mouse centrin1 has been crystallized. The mouse centrin1 has been expressed in Escherichia coli as a GST‐centrin fusion protein containing a thrombin protease cleavage site between the fusion partners. Two constructs with different linking‐sequence lengths were expressed and purified. Thrombin cleavage yielded functional centrin1 and N‐terminally extended centrin1 containing 25 additional residues upstream of its N‐terminus. Only N‐terminally extended centrin1 (MW ≃ 22 240 Da) could be crystallized at room temperature, using 20–25%(w/v) PEG 1500, 5–10%(v/v) ethylene glycol and 1–2%(v/v) dioxane. Crystals were suitable for X‐ray analysis, diffracting to 2.9 Å at 295 K using a rotating‐anode X‐ray source. They belong to space group C2, with unit‐cell parameters a = 60.7, b = 59.6, c = 58.3 Å, β = 109.4°. Assuming the asymmetric cell to be occupied by one centrin1 molecule of 22.2 kDa, the unit cell contains 45% solvent with a crystal volume per protein weight, VM, of 2.2 Å3 Da−1.  相似文献   

19.
    
We have previously described a recombinase‐mediated gene stacking system in which the Cre recombinase is used to remove lox‐site flanked DNA no longer needed after each round of Bxb1 integrase‐mediated site‐specific integration. The Cre recombinase can be conveniently introduced by hybridization with a cre‐expressing plant. However, maintaining an efficient cre‐expressing line over many generations can be a problem, as high production of this DNA‐binding protein might interfere with normal chromosome activities. To counter this selection against high Cre activity, we considered a split‐cre approach, in which Cre activity is reconstituted after separate parts of Cre are brought into the same genome by hybridization. To insure that the recombinase‐mediated gene stacking system retains its freedom to operate, we tested for new locations to split Cre into complementing fragments. In this study, we describe testing four new locations for splitting the Cre recombinase for protein fragment complementation and show that the two fragments of Cre split between Lys244 and Asn245 can reconstitute activity that is comparable to that of wild‐type Cre.  相似文献   

20.
    
S100A13 is a member of the S100 family of EF‐hand‐containing calcium‐binding proteins and plays an important role in the secretion of fibroblast growth factor‐1 and interleukin 1α, two pro‐angiogenic factors released by the endoplasmic reticulum/Golgi‐independent non‐classical secretory pathway. Human S100A13 was heterologously expressed in Escherichia coli, purified and crystallized by the hanging‐drop vapour‐diffusion method using PEG 3350 as the precipitant. The crystals diffracted X‐rays from a synchrotron‐radiation source to 1.8 Å resolution and the space group was assigned as primitive orthorhombic P212121.  相似文献   

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