Measuring how two proteins affect each other's net charge in a crowded environment
Measuring how two proteins affect each other's net charge in a crowded environment
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DOI：
10.1002/pro.4092
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发表时间：
2021-05-12
期刊：
影响因子：
8
通讯作者：
Shaw, Bryan F.
中科院分区：
文献类型：
--
作者：
Dashnaw, Chad M.;Koone, Jordan C.;Shaw, Bryan F.
理论预测，蛋白质的净电荷（Z）可以被相邻蛋白质的净电荷改变，因为两者在德拜长度以下相互接近。这种类型的电荷调节表明，蛋白质的电荷和功能可能会受到邻近蛋白质的影响，而不会直接结合。由于分析挑战，蛋白质拥挤期间的电荷调节从未被直接测量。在这里，我们表明，赖氨酸特异性蛋白质交联剂（NHS酯-施陶丁格对）可用于模拟拥挤，通过连接两个非相互作用的蛋白质在最大距离相似的7.9埃。然后可以用赖氨酸-酰基“蛋白质电荷梯”和毛细管电泳测定区域异构二聚体和前单体的净电荷。作为概念证明，我们共价连接肌红蛋白（Z（单体）= -0.43 +/-0.01）和α-乳白蛋白（Z（单体）= -4.63 +/-0.05）。酰胺氢/氘交换和圆二色性光谱表明，交联没有显着改变蛋白质的结构或导致直接结合（从而模仿拥挤）。最后，二聚体电荷梯的毛细管电泳分析检测到在被该对拥挤时Δ Z = -0.04 +/-0.09的电荷变化（Z（二聚体）= -5.10 +/-0.07）。这些小的Δ Z值不一定是一般的蛋白质拥挤（定性或定量），但将根据蛋白质大小，电荷和溶剂条件而变化。
Theory predicts that the net charge (Z) of a protein can be altered by the net charge of a neighboring protein as the two approach one another below the Debye length. This type of charge regulation suggests that a protein's charge and perhaps function might be affected by neighboring proteins without direct binding. Charge regulation during protein crowding has never been directly measured due to analytical challenges. Here, we show that lysine specific protein crosslinkers (NHS ester-Staudinger pairs) can be used to mimic crowding by linking two non-interacting proteins at a maximal distance of similar to 7.9 angstrom. The net charge of the regioisomeric dimers and preceding monomers can then be determined with lysine-acyl "protein charge ladders" and capillary electrophoresis. As a proof of concept, we covalently linked myoglobin (Z(monomer) = -0.43 +/- 0.01) and alpha-lactalbumin (Z(monomer) = -4.63 +/- 0.05). Amide hydrogen/deuterium exchange and circular dichroism spectroscopy demonstrated that crosslinking did not significantly alter the structure of either protein or result in direct binding (thus mimicking crowding). Ultimately, capillary electrophoretic analysis of the dimeric charge ladder detected a change in charge of Delta Z = -0.04 +/- 0.09 upon crowding by this pair (Z(dimer) = -5.10 +/- 0.07). These small values of Delta Z are not necessarily general to protein crowding (qualitatively or quantitatively) but will vary per protein size, charge, and solvent conditions.