Mapping local protein electrostatics by EPR of pH-sensitive thiol-specific nitroxide.
Mapping local protein electrostatics by EPR of pH-sensitive thiol-specific nitroxide.
复制标题
通过 pH 敏感的硫醇特异性硝基氧的 EPR 绘制局部蛋白质静电图。
DOI：
10.1021/bi800272f
复制
发表时间：
2008
期刊：
影响因子：
2.9
通讯作者：
Smirnov,AlexI
中科院分区：
文献类型：
--
作者：
Voinov,MaximA;Ruuge,Andres;Reznikov,VladimirA;Grigor'ev,IgorA;Smirnov,AlexI
合成并表征了咪唑烷系列中首个巯基ph敏感的硝基自旋标记物甲基乙硫磺酸-（1-氧-2,2,3,5,5-五甲基咪唑烷-4-基甲基）酯（IMTSL）。研究了新自旋标签自由形态下与氨基酸半胱氨酸和谷胱甘肽共价连接的x波段（9 GHz）和w波段（94 GHz） EPR光谱参数与pH和溶剂极性的关系。发现自旋标记的可质子化叔氨基的pkvalue不受存在于非结构小肽侧链中的其他可电离基团的影响。w波段EPR光谱被证明可以通过精确的因子测量来测定钾。已经证明，无论测量频率或自旋交换机制如何，对ph敏感的氮氧化物的pka测定都可以达到高精度：在x波段快，在w波段慢。研究发现，IMTSL与一种来自酵母的模型蛋白iso1 - cytochromece发生特异性反应，其EPR光谱与最常用的半胱氨酸特异性标记MTSL非常相似。CD数据显示，在IMTSL标记后，蛋白质的整体结构没有受到扰动。结果表明，对于IMTSL，gisoco与aiso呈线性相关，但对于中性形式和带电形式的氮氧化物，其斜率不同。这一发现归因于溶剂对NO基团氧原子自旋密度和氧孤对轨道激发能的影响。
A first thiol-specific pH-sensitive nitroxide spin-label of the imidazolidine series, methanethiosulfonic acidS-(1-oxyl-2,2,3,5,5-pentamethylimidazolidin-4-ylmethyl) ester (IMTSL), has been synthesized and characterized. X-Band (9 GHz) and W-band (94 GHz) EPR spectral parameters of the new spin-label in its free form and covalently attached to an amino acid cysteine and a tripeptide glutathione were studied as a function of pH and solvent polarity. The pKavalue of the protonatable tertiary amino group of the spin-label was found to be unaffected by other ionizable groups present in side chains of unstructured small peptides. The W-band EPR spectra were shown to allow for pKadetermination from preciseg-factor measurements. Is has been demonstrated that the high accuracy of pKadetermination for pH-sensitive nitroxides could be achieved regardless of the frequency of measurements or the regime of spin exchange: fast at X-band and slow at W-band. IMTSL was found to react specifically with a model protein, iso-1-cytochromecfrom the yeastSaccharomyces cerevisiae, giving EPR spectra very similar to those of the most commonly employed cysteine-specific label MTSL. CD data indicated no perturbations to the overall protein structure upon IMTSL labeling. It was found that for IMTSL,gisocorrelates linearly withAiso, but the slopes are different for the neutral and charged forms of the nitroxide. This finding was attributed to the solvent effects on the spin density at the oxygen atom of the NO group and on the excitation energy of the oxygen lone-pair orbital.