排序方式: 共有14条查询结果,搜索用时 125 毫秒
11.
Magnesium is essential for all forms of life. It is the cofactor for many enzymes and plays a key role in many biological processes. Thus, the acquisition of Mg2+ is crucial for cell survival. The best characterized Mg2+ transporters to date belong to the 2-TM-GxN type family of transporters. The name indicates the two C-terminal transmembrane (TM) domains and a conserved GxN motif present in all members of this family towards the C-terminal end of TM1. In most members of the family, this conserved motif is generally YGMNF. The prototypical member of this family is CorA. Other characterized members of this family include Mrs2p, Alr, Mnr, AtMGT and ZntB. CorA is widely distributed throughout the prokaryotic world. It is the primary Mg2+ uptake system in most bacteria and many Archaea. A homolog, Mrs2p, is a eukaryotic mitochondrial Mg2+ channel. The Mrs2p related AtMGT transporters are found in plants and other eukaryotes. Alr1p and Mnr are Mg2+ transporters found in the plasma membrane of many fungi. ZntB is a bacterial member of the 2-TM-GxN family but mediates efflux of Zn2+ instead of influx of Mg2+. The recent crystal structure of a bacterial CorA shows that the structure of this family is unlike that of any other class of transporter or channel currently known. 相似文献
12.
R. M. Cleverley M. Saleem J. Kean R. C. Ford J. P. Derrick S. M. Prince 《Molecular membrane biology》2013,30(8):625-630
A method to rapidly assess the oligomeric composition of multimeric proteins is notably absent from reported schemes for high throughput production and crystallization of membrane proteins. In this report we have investigated the suitability of PFO-PAGE electrophoresis for this purpose and present examples where it proves highly informative in selecting conditions favouring the functional oligomeric state of the target protein. Features such as the ability to analyze several samples in parallel, including crude membrane extracts, suggest it will be highly adaptable to high throughput analysis of membrane proteins. 相似文献
13.
Due to their high hydrophobicity, it is a challenge to obtain high yields of transmembrane peptides for structural and functional characterization. In the present work, a robust method is developed for the expression, purification and reconstitution of transmembrane peptides, especially for those containing conserved methionines. By using a truncated and mutated glutathione-S-transferase construct as the carrier protein and hydroxylamine (which specifically cleaves the peptide bond between Asn and Gly) as the cleavage reagent, 10 mg of the first transmembrane helix of CorA, a Mg2+ transporter from Mycobacterium tuberculosis, can be conveniently obtained with high purity from 1 L of M9 minimal media under optimized conditions. The biophysical properties of the peptide were studied by circular dichroism and nuclear magnetic resonance spectroscopy, and the results show that this CorA peptide is well folded in detergent micelles and the secondary structure is very similar to that in recent crystal structures. In addition, this CorA construct is oligomeric in perfluoro-octanoic acid micelles. The compatibility with the transmembrane peptides containing conserved methionines, the high yield and the simple process make the present method competitive with other commonly used methods to produce such peptides for structural and functional studies. 相似文献
14.
《Journal of molecular biology》2023,435(17):168192
CorA, the primary magnesium ion channel in prokaryotes and archaea, is a prototypical homopentameric ion channel that undergoes ion-dependent conformational transitions. CorA adopts five-fold symmetric non-conductive states in the presence of high concentrations of Mg2+, and highly asymmetric flexible states in its complete absence. However, the latter were of insufficient resolution to be thoroughly characterized. In order to gain additional insights into the relationship between asymmetry and channel activation, we exploited phage display selection strategies to generate conformation-specific synthetic antibodies (sABs) against CorA in the absence of Mg2+. Two sABs from these selections, C12 and C18, showed different degrees of Mg2+-sensitivity. Through structural, biochemical, and biophysical characterization, we found the sABs are both conformation-specific but probe different features of the channel under open-like conditions. C18 is highly specific to the Mg2+-depleted state of CorA and through negative-stain electron microscopy (ns-EM), we show sAB binding reflects the asymmetric arrangement of CorA protomers in Mg2+-depleted conditions. We used X-ray crystallography to determine a structure at 2.0 Å resolution of sAB C12 bound to the soluble N-terminal regulatory domain of CorA. The structure shows C12 is a competitive inhibitor of regulatory magnesium binding through its interaction with the divalent cation sensing site. We subsequently exploited this relationship to capture and visualize asymmetric CorA states in different [Mg2+] using ns-EM. We additionally utilized these sABs to provide insights into the energy landscape that governs the ion-dependent conformational transitions of CorA. 相似文献