Ramalingam T S, Das P K, Podder S K
Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore.
Biochemistry. 1994 Oct 11;33(40):12247-54. doi: 10.1021/bi00206a030.
The entry of the plant toxin ricin and its A- and B-subunits in model membranes in the presence as well as absence of monosialoganglioside (GM1) has been studied. Dioleoylphosphatidylcholine and 5-, 10-, and 12-doxyl- or 9,10-dibromophosphatidylcholines serve as quenchers of intrinsic tryptophan fluorescence of the proteins. The parallax method of Chattopadhyay and London [(1987) Biochemistry 26, 39-45] has been employed to measure the average membrane penetration depth of tryptophans of ricin and its B-chain and the actual depth of the sole Trp 211 in the A-chain. The results indicate that both of the chains as well as intact ricin penetrate the membrane deeply and the C-terminal end of the A-chain is well inside the bilayer, especially at pH 4.5. An extrinsic probe N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (I-AEDANS) has been attached to Cys 259 of the A-chain, and the kinetics of penetration has been followed by monitoring the increase in AEDANS fluorescence at 480 nm. The insertion follows first-order kinetics, and the rate constant is higher at a lower pH. The energy transfer distance analysis between Trp 211 and AEDANS points out that the conformation of the A-chain changes as it inserts into the membrane. CD studies indicate that the helicity of the proteins increases after penetration, which implies that some of the unordered structure in the native protein is converted to the ordered form during this process. Hydrophobic forces seem to be responsible for stabilizing a particular protein conformation inside the membrane.
研究了在存在和不存在单唾液酸神经节苷脂（GM1）的情况下，植物毒素蓖麻毒素及其A和B亚基进入模型膜的情况。二油酰磷脂酰胆碱和5-、10-和12-二氧代-或9,10-二溴磷脂酰胆碱用作蛋白质内在色氨酸荧光的猝灭剂。采用Chattopadhyay和London [(1987) Biochemistry 26, 39 - 45]的视差法来测量蓖麻毒素及其B链色氨酸的平均膜穿透深度以及A链中唯一色氨酸Trp 211的实际深度。结果表明，两条链以及完整的蓖麻毒素都能深入穿透膜，A链的C末端很好地位于双层膜内部，尤其是在pH 4.5时。一种外在探针N-(碘乙酰)-N'-(5-磺基-1-萘基)乙二胺（I-AEDANS）已连接到A链的Cys 259上，并通过监测480 nm处AEDANS荧光的增加来跟踪穿透动力学。插入遵循一级动力学，且在较低pH下速率常数更高。Trp 211与AEDANS之间的能量转移距离分析表明，A链在插入膜时其构象会发生变化。圆二色性（CD）研究表明，蛋白质穿透后螺旋度增加，这意味着天然蛋白质中的一些无序结构在此过程中会转变为有序形式。疏水力似乎负责稳定膜内特定的蛋白质构象。
