Nyitrai M, Hild G, Lakos Z, Somogyi B
Research Group of the Hungarian Academy of Sciences at the Department of Biophysics, University Medical School of Pécs, Hungary.
Biophys J. 1998 May;74(5):2474-81. doi: 10.1016/S0006-3495(98)77955-X.
A fluorescence resonance energy transfer (FRET) parameter, f' (defined as the average transfer efficiency, (E), normalized by the actual fluorescence intensity of the donor in the presence of acceptor, F(DA)), was previously shown to be capable of monitoring both changes in local flexibility of the protein matrix and major conformational transitions. The temperature profile of this parameter was used to detect the change of the protein flexibility in the small domain of the actin monomer (G-actin) upon the replacement of Ca2+ by Mg2+. The Cys-374 residue of the actin monomer was labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) to introduce a fluorescence donor and the Lys-61 residue with fluorescein-5-isothiocyanate (FITC) to serve as an acceptor. The f' increases with increasing temperature over the whole temperature range for Mg-G-actin. This parameter increases similarly in the case of Ca-G-actin up to 26 degrees C, whereas an opposite tendency appears above this temperature. These data indicate that there is a conformational change in Ca-G-actin above 26 degrees C that was not detected in the case of Mg-G-actin. In the temperature range between 6 degrees C and 26 degrees C the slope of the temperature profile of f' is the same for Ca-G-actin and Mg-G-actin, suggesting that the flexibility of the protein matrix between the two labels is identical in the two forms of actin.
一种荧光共振能量转移（FRET）参数f′（定义为平均转移效率（E），通过供体在存在受体时的实际荧光强度F(DA)进行归一化），先前已表明能够监测蛋白质基质局部柔韧性的变化以及主要的构象转变。该参数的温度曲线用于检测肌动蛋白单体（G-肌动蛋白）小结构域中蛋白质柔韧性在Ca2+被Mg2+取代时的变化。肌动蛋白单体的Cys-374残基用N-碘乙酰基-N′-(5-磺基-1-萘基)乙二胺（IAEDANS）标记以引入荧光供体，而Lys-61残基用异硫氰酸荧光素（FITC）标记以作为受体。对于Mg-G-肌动蛋白，在整个温度范围内f′随温度升高而增加。在Ca-G-肌动蛋白中，该参数在高达26℃时同样增加，而在该温度以上则出现相反的趋势。这些数据表明，Ca-G-肌动蛋白在26℃以上存在构象变化，而Mg-G-肌动蛋白中未检测到这种变化。在6℃至26℃的温度范围内，Ca-G-肌动蛋白和Mg-G-肌动蛋白的f′温度曲线斜率相同，这表明两种形式的肌动蛋白中两个标记之间蛋白质基质的柔韧性是相同的。
