Guzman Irisbel, Ghaemi Zhaleh, Baranger Anne, Luthey-Schulten Zaida, Gruebele Martin
Department of Biochemistry, and ‡Department of Chemistry, Department of Physics, Center for the Physics of Living Cells, and Center for Biophysics and Quantitative Biology, University of Illinois , Urbana, Illinois 61801, United States.
J Phys Chem B. 2015 Mar 5;119(9):3651-61. doi: 10.1021/jp511760m. Epub 2015 Feb 24.
The complex of spliceosomal U1A protein and its cognate SL2 RNA is a prototype system for protein-RNA binding studies. A major question is whether U1A protein alone is capable of undergoing conformational dynamics similar to structural rearrangements upon RNA binding. Using a fast temperature jump and tryptophan fluorescence detection, we uncover a ∼20 μs conformational transition for the Lys22Gln/Phe56Trp-only mutant of U1A, yet a Phe56Trp-only control mutant does not show the transition. To explain this observation, we performed extensive molecular dynamics (MD) simulations. The simulations explain why only the Lys22Gln/Phe56Trp-only mutant shows a fluorescence signal: in the other mutant, the tryptophan probe is not quenched upon structural rearrangement. The simulations support helix C movement as the underlying structural rearrangement, although the simulated time scale is faster than experimentally detected. On the basis of our MD results, we propose a reversible two-pathway three-state transition for the helix C movement and assign T-jump kinetics to a closed to semi-closed transition of the helix. Our result provides a specific example of how alternative protein conformations on the native side of the folding barrier can be functionally important, for example in conformational selection by a binding partner.
剪接体U1A蛋白及其同源SL2 RNA的复合物是蛋白质-RNA结合研究的一个原型系统。一个主要问题是，单独的U1A蛋白是否能够经历类似于RNA结合时结构重排的构象动力学变化。我们使用快速温度跳跃和色氨酸荧光检测，发现U1A的仅Lys22Gln/Phe56Trp突变体存在约20微秒的构象转变，而仅Phe56Trp对照突变体则未显示出这种转变。为了解释这一观察结果，我们进行了广泛的分子动力学（MD）模拟。这些模拟解释了为什么只有仅Lys22Gln/Phe56Trp突变体显示出荧光信号：在另一个突变体中，色氨酸探针在结构重排时未被淬灭。模拟结果支持螺旋C的移动是潜在的结构重排，尽管模拟的时间尺度比实验检测到的要快。基于我们的MD结果，我们提出了螺旋C移动的可逆双途径三态转变，并将温度跳跃动力学归因于螺旋从封闭到半封闭的转变。我们的结果提供了一个具体例子，说明折叠屏障天然侧的替代蛋白构象如何在功能上具有重要意义，例如在结合伴侣的构象选择中。
