Schröder Gunnar F, Alexiev Ulrike, Grubmüller Helmut
Theoretical and Computational Biophysics Department, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany.
Biophys J. 2005 Dec;89(6):3757-70. doi: 10.1529/biophysj.105.069500. Epub 2005 Sep 16.
Time-resolved fluorescence anisotropy decay experiments on a protein-attached dye can probe local protein dynamics and steric restrictions, but are difficult to interpret at the structural level. Aiming at an atomistic description, we have carried out molecular dynamics simulations of such experiments. Our simulations describe an Alexa488 fluorescent dye maleimide derivative covalently attached via a single cysteine to the AB-loop of bacteriorhodopsin. Fluorescence anisotropy decay curves obtained from the simulations agree well with the measured ones. Three anisotropy decay components were resolved and assigned to: 1), the fast dynamics of the attached dye on the picosecond timescale; 2), the slower dynamics of the loop at the one nanosecond timescale; and 3), the overall tumbling of the molecule. For the biologically relevant 1-ns component we identified two processes from simulations, the motion of the flexible loop as well as slow conformational dynamics of the dye. These two processes are not separable by experiment alone. Furthermore, analysis of the correlation between the dye and the protein motion revealed which part and which motion of the protein is actually probed by the experiment. Finally, our simulations allowed us to test the usual and inevitable assumption underlying these types of spectroscopic measurements that the attached dye probe does not severely perturb the protein dynamics. For the case at hand, by comparison with a simulation of the dye-free protein, the perturbation was quantified and found to be small.
对附着有染料的蛋白质进行时间分辨荧光各向异性衰减实验，能够探测蛋白质的局部动力学和空间位阻限制，但在结构层面上却难以解读。为了实现原子层面的描述，我们开展了此类实验的分子动力学模拟。我们的模拟描述了一种Alexa488荧光染料马来酰亚胺衍生物，它通过单个半胱氨酸共价连接到细菌视紫红质的AB环上。模拟得到的荧光各向异性衰减曲线与实测曲线吻合良好。分辨出了三个各向异性衰减成分并将其归因于：1）附着染料在皮秒时间尺度上的快速动力学；2）环在一纳秒时间尺度上的较慢动力学；3）分子的整体翻滚。对于生物学上相关的1纳秒成分，我们从模拟中识别出两个过程，即柔性环的运动以及染料的缓慢构象动力学。这两个过程无法仅通过实验区分开来。此外，对染料与蛋白质运动之间相关性的分析揭示了实验实际探测的是蛋白质的哪一部分以及哪种运动。最后，我们的模拟使我们能够检验这些类型光谱测量所基于的通常且不可避免的假设，即附着的染料探针不会严重干扰蛋白质动力学。对于当前的情况，通过与无染料蛋白质的模拟进行比较，对这种干扰进行了量化，发现其很小。
