Kardos Roland, Nevalainen Elisa, Nyitrai Miklós, Hild Gábor
University of Pécs, Medical School, Department of Biophysics, Szigeti str. 12, Pécs H-7624, Hungary; Szentágothai Research Center, Ifjúság str. 34, Pécs H-7624, Hungary.
Biochim Biophys Acta. 2013 Oct;1834(10):2010-9. doi: 10.1016/j.bbapap.2013.06.006. Epub 2013 Jul 8.
The main goal of the work was to uncover the dynamical changes in actin induced by the binding of cofilin and profilin. The change in the structure and flexibility of the small domain and its function in the thermodynamic stability of the actin monomer were examined with fluorescence spectroscopy and differential scanning calorimetry (DSC). The structure around the C-terminus of actin is slightly affected by the presence of cofilin and profilin. Temperature dependent fluorescence resonance energy transfer measurements indicated that both actin binding proteins decreased the flexibility of the protein matrix between the subdomains 1 and 2. Time resolved anisotropy decay measurements supported the idea that cofilin and profilin changed similarly the dynamics around the fluorescently labeled Cys-374 and Lys-61 residues in subdomains 1 and 2, respectively. DSC experiments indicated that the thermodynamic stability of actin increased by cofilin and decreased in the presence of profilin. Based on the information obtained it is possible to conclude that while the small domain of actin acts uniformly in the presence of cofilin and profilin the overall stability of actin changes differently in the presence of the studied actin binding proteins. The results support the idea that the small domain of actin behaves as a rigid unit during the opening and closing of the nucleotide binding pocket in the presence of profilin and cofilin as well. The structural arrangement of the nucleotide binding cleft mainly influences the global stability of actin while the dynamics of the different segments can change autonomously.
这项工作的主要目标是揭示肌动蛋白丝切蛋白和肌动蛋白解聚因子结合所诱导的肌动蛋白动力学变化。利用荧光光谱和差示扫描量热法（DSC）研究了小结构域的结构和柔韧性变化及其在肌动蛋白单体热力学稳定性中的作用。肌动蛋白丝切蛋白和肌动蛋白解聚因子的存在对肌动蛋白C末端周围的结构有轻微影响。温度依赖性荧光共振能量转移测量表明，这两种肌动蛋白结合蛋白均降低了1和2亚结构域之间蛋白质基质的柔韧性。时间分辨各向异性衰减测量结果支持以下观点：肌动蛋白丝切蛋白和肌动蛋白解聚因子分别对1和2亚结构域中荧光标记的Cys-374和Lys-61残基周围的动力学产生类似的变化。DSC实验表明，肌动蛋白丝切蛋白可提高肌动蛋白的热力学稳定性，而肌动蛋白解聚因子则会降低其稳定性。根据所获得的信息可以得出结论，虽然在肌动蛋白丝切蛋白和肌动蛋白解聚因子存在的情况下，肌动蛋白的小结构域作用一致，但在研究的肌动蛋白结合蛋白存在的情况下，肌动蛋白的整体稳定性变化不同。结果支持这样的观点：在肌动蛋白解聚因子和肌动蛋白丝切蛋白存在的情况下，肌动蛋白的小结构域在核苷酸结合口袋打开和关闭过程中表现为一个刚性单元。核苷酸结合裂隙的结构排列主要影响肌动蛋白的整体稳定性，而不同片段的动力学可以自主变化。
