Interaction of anesthetics with macromolecules

描述(根据申请者的申请改编)：吸入性麻醉剂是目前医生使用的毒性最大、了解最少的药物。只有通过详细了解它们的结合相互作用、位点和目标，才能实现改进。在该计划的这个子项目中，我们将继续使用等温滴定热法和酰胺氢交换(目标1)将候选蛋白质靶标的结构特征与吸入麻醉剂结合的化学计量和能量学联系起来。每个靶在结构上都有定义，因此空穴数量、形状、体积和极性等特征可以与束缚能量学相关联。这些特征将被参数化为一个预测性的麻醉剂结合算法，使用现有的高分辨率载脂蛋白和血清白蛋白复合体进行训练，以从蛋白质数据库和蛋白质结构倡议中挖掘进一步的潜在目标。在目标2中，来自目标1的显示特异性结合的蛋白质将与各种吸入麻醉剂共结晶，以获得进一步的结构细节，将其并入结合算法，用于亲和力的相关分析，并探索结合的结构后果。除了载脂蛋白和血清白蛋白外，在最后一个周期中发现了4种蛋白质，将进行初步检测。最后，在目标3中，麻醉剂光标记这一强大的方法将从两个方面进一步完善。首先，结合光标记法和结晶学将允许验证揭示的位置，以及对结合的选择性和机制的估计。将继续开发重氮和重氮挥发性化合物，并将重点放在加合检测方法上。其他4个项目中的每一个都将存在重要的相互作用；这些目标将通过揭示亲和力和化学计量学的基础-改变蛋白质构象的能量驱动因素-以及揭示新的和意想不到的吸入麻醉蛋白质靶标-无论是通过挖掘现有数据库，还是通过光标记识别-来为整个计划目标做出贡献。

DESCRIPTION (adapted from applicant's application): Inhaled anesthetics are the most toxic and least understood drugs that physicians currently use. Improvements can only occur via a detailed understanding of their binding interactions, sites and targets. In this subproject of the program, we will continue to relate structural features of candidate protein targets with the stoichiometry and energetics of inhaled anesthetic binding using both isothermal titration calorimetry and amide hydrogen exchange (aim 1). Each target is structurally defined, so that features, such as cavity number, shape, volume, and polarity can be correlated with binding energetics. These features will be parameterized into a predictive, anesthetic binding algorithm, using the existing high-resolution apoferritin and serum albumin complexes for training, to mine further potential targets from the Protein Data Bank and the Protein Structure Initiative. In aim 2, proteins from aim 1 that show specific binding will be co-crystallized with various inhaled anesthetics to gain further structural detail to be incorporated into the binding algorithm, to use in correlative analyses of affinity, and to probe for the structural consequences of binding. In addition to apoferritin and serum albumin, 4 proteins were identified in the last cycle that will be examined initially. Finally, in aim 3, the powerful method of anesthetic photolabeling will be further refined in two ways. First, combining photolabeling with crystallography will allow validation of revealed location, and an estimate of selectivity and mechanism of incorporation. Continued development of diazirine and diazo volatile compounds will occur, and emphasis will be placed on adduction detection methods. Important interactions will each of the other 4 projects exist; these aims will contribute to the overall program goals by revealing the basis for affinity and stoichiometry- the energetic driver for altering protein conformation, and by revealing new and unanticipated inhaled anesthetic protein targets - either from the mining of existing databases, or through identification via photolabeling.