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引用次数: 0
摘要
Na+、K+-ATPase(NKA)的核苷酸结合域(N-domain)的理化特征是含有大量的 Glu 和 Asp 残基,因此等电点较低(pI = 5.0)。众所周知,酸性蛋白质会与阳离子相互作用。硅学分析揭示了 NKA N 域结构中潜在的阳离子相互作用位点。使用重组的 NKA N-domain 对其与阳离子的相互作用进行了体外测试。经丙烯酰胺介导的荧光淬灭测定,N-domain 蛋白表面含有两个 Trp 残基,可通过荧光变化进行结构研究。阳离子(Na+、K+、Ca2+)的存在会降低 N-结构域的本征荧光,这表明阳离子对蛋白质结构有影响。ATP 结合也会降低 N-结构域的本征荧光,从而可以确定核苷酸的亲和性。在阳离子存在的情况下,N-结构域对 ATP 的亲和力增加。异硫氰酸荧光素(FITC)与 N-domain 的分子对接显示了两种结合模式,异硫氰酸基位于核苷酸结合位点 Lys485 和 Lys506 附近 5-6 Å 处。ATP 的存在阻止了 N-domain 的 FITC 共价标记,这表明该结合位点存在竞争行为。据推测,阳离子与 N-domain 结构相互作用,从而调节核苷酸(ATP)的亲和力,并可能影响 NKA 的催化作用。
Effect of Cations on ATP Binding to the N-domain of Na+, K+-ATPase
The nucleotide-binding domain (N-domain) of the Na+, K+-ATPase (NKA) is physicochemically characterized by a high content of Glu and Asp residues, resulting in a low isoelectric point (pI = 5.0). Acidic proteins are known to interact with cations. The analysis in silico revealed potential cation interaction sites in the NKA N-domain structure. The interaction with cations was tested in vitro by using a recombinant NKA N-domain. The N-domain contains two Trp residues at the protein surface, as determined by acrylamide-mediated fluorescence quenching, that are useful for structural studies through fluorescence changes. Intrinsic fluorescence of the N-domain was decreased by the presence of cations (Na+, K+, Ca2+) indicating an effect on the protein structure. ATP binding also decreased the N-domain intrinsic fluorescence, which allowed nucleotide affinity determination. In the presence of cations, the N-domain affinity for ATP was increased. Molecular docking of fluorescein isothiocyanate (FITC) with the N-domain showed two binding modes with the isothiocyanate group located 5–6 Å close to Lys485 and Lys506 in the nucleotide-binding site. The presence of ATP prevented the FITC covalent labeling of the N-domain demonstrating the competitive behavior for the binding site. It is proposed that cations interact with the N-domain structure and thereby modulate nucleotide (ATP) affinity and possibly affecting NKA catalysis.
期刊介绍:
Journal of Fluorescence is an international forum for the publication of peer-reviewed original articles that advance the practice of this established spectroscopic technique. Topics covered include advances in theory/and or data analysis, studies of the photophysics of aromatic molecules, solvent, and environmental effects, development of stationary or time-resolved measurements, advances in fluorescence microscopy, imaging, photobleaching/recovery measurements, and/or phosphorescence for studies of cell biology, chemical biology and the advanced uses of fluorescence in flow cytometry/analysis, immunology, high throughput screening/drug discovery, DNA sequencing/arrays, genomics and proteomics. Typical applications might include studies of macromolecular dynamics and conformation, intracellular chemistry, and gene expression. The journal also publishes papers that describe the synthesis and characterization of new fluorophores, particularly those displaying unique sensitivities and/or optical properties. In addition to original articles, the Journal also publishes reviews, rapid communications, short communications, letters to the editor, topical news articles, and technical and design notes.