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EPR assessment of protein sites for incorporation of Gd(III) MRI contrast labels

机译：结合Gd（III）MRI对比标签的蛋白质位点的EPR评估

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We have engineered apolipoprotein Aa??I (apoAa??I), a major protein constituent of higha??density lipoprotein (HDL), to contain DOTAa??chelated Gd(III) as an MRI contrast agent for the purpose of imaging reconstituted HDL (rHDL) biodistribution, metabolism and regulation in vivo. This protein contrast agent was obtained by attaching the thiola??reactive Gd[MTSa??ADO3A] label at Cys residues replaced at four distinct positions (52, 55, 76 and 80) in apoAa??I. MRI of infused mice previously showed that the Gda??labeled apoAa??I migrates to both the liver and the kidney, the organs responsible for HDL catabolism; however, the contrast properties of apoAa??I are superior when the ADO3A moiety is located at position 55, compared with the protein labeled at positions 52, 76 or 80. It is shown here that continuous wave Xa??band (9a??GHz) electron paramagnetic resonance (EPR) spectroscopy is capable of detecting differences in the Gd(III) signal when comparing the labeled protein in the lipida??free with the rHDL state. Furthermore, the values of NMR relaxivity obtained for labeled variants in both the lipida??free and rHDL states correlate to the product of the Xa??band Gd(III) spectral width and the collision frequency between a nitroxide spin label and a polar relaxation agent. Consistent with its superior relaxivity measured by NMR, the rHDLa??associated apoAa??I containing the Gd[MTSa??ADO3A] probe attached to position 55 displays favorable dynamic and water accessibility properties as determined by Xa??band EPR. While room temperature EPR requires 1a??m m Gd(III)a??labeled and only 10a???μ m nitroxidea??labeled protein to resolve the spectrum, the volume requirement is exceptionally low (~5a???μl). Thus, Xa??band EPR provides a practical assessment for the suitability of imaging candidates containing the sitea??directed ADO3A contrast probe. Copyright ?? 2013 John Wiley & Sons, Ltd.
机译：我们已经设计了载脂蛋白Aa ?? I（apoAa ?? I），它是高密度脂蛋白（HDL）的主要蛋白质成分，其中含有DOTAa ??螯合的Gd（III）作为MRI造影剂，用于重组成像。 HDL（rHDL）在体内的生物分布，代谢和调节。该蛋白造影剂是通过将硫醇-α反应性的Gd [MTSa-ADO3A]标记附着于在apoAaβI的四个不同位置（52、55、76和80​​）处置换的Cys残基上而获得的。输注小鼠的MRI以前显示，Gdaα标记的apoAaβⅠ迁移到肝脏和肾脏，这是导致HDL分解代谢的器官。然而，与标记在位置52、76或80的蛋白质相比，当ADO3A部分位于位置55时，apoAaΔII的对比度特性是优异的。当将无脂质α-标记的蛋白质与rHDL状态进行比较时，GHz顺磁共振电子波谱法能够检测Gd（III）信号的差异。此外，在无脂质和无rHDL两种状态下，为标记变体获得的NMR弛豫度值与Xaδ带Gd（III）谱宽和一氧化氮自旋标记与极性弛豫之间的碰撞频率的乘积相关。代理商。与通过NMR测得的其优异的弛豫性相一致，含有连接到位置55的Gd [MTSa-ADO3A]探针的rHDLa 13-相关的载脂蛋白A -I显示出良好的动态和水可及性，这是通过Xa-能带EPR测定的。室温EPR需要> 1a ?? mm Gd（III）a ??标记的蛋白，而仅需> 10a？mmμm的亚硝酸根??标记的蛋白来解析光谱，而体积要求却异常低（〜5a？μlµl ）。因此，Xa 1带EPR为包含定点定向的ADO3A对比探针的成像候选物的适用性提供了实用的评估。版权?? 2013 John Wiley＆Sons，Ltd.

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