Watching proteins fold one at a time

生命科学中最大的挑战之一是理解蛋白质的氨基酸序列与其三维形状之间的复杂关系。蛋白质折叠是如此复杂，因为未折叠的多肽可以通过无数种方式到达功能折叠结构。由于避免了平均化，单分子方法特别适合于跟踪单独的折叠路径，测试和改进当前的模型。我的研究计划将使用一种创新的荧光显微镜，它可以同时记录每个光子的波长、发射时间和偏振。可以同时跟踪多个分子参数的演化，通过共振能量转移(FRET)测量距离的精度将得到提高。我将在有利于在折叠和未折叠状态之间频繁转换的条件下研究不同大小、来源和功能的蛋白质(S)。折叠的时间进程将使用FRET和新的环境敏感探针相结合来检测。在细胞中，蛋白质折叠可能开始于肽合成的非常早期阶段，沿着横跨核糖体的狭窄隧道，但特征动力学和构象变化仍然难以捉摸。预计多参数单分子研究将对新生多肽的折叠动力学、与核糖体以及与辅助生物分子(伴侣)的相互作用提供独特的见解。

One of the greatest challenges in life sciences is to understand the complex relationship between the amino acid sequence of a protein and its three-dimensional shape. Protein folding is so complex because of countless ways an unfolded peptide can reach the functional folded structure. By avoiding averaging, single-molecule methods are uniquely suited to follow individual folding pathways, test and improve the current models. My research program will make use of an innovative fluorescence microscope that records simultaneously the wavelength, the emission time and the polarization of each photon. The evolution of several molecular parameters can be tracked simultaneously and the accuracy of distance measurements through resonance energy transfer (FRET) will be improved. I will study proteins of different size, origin and function under conditions that favor frequent transitions between the folded and the unfolded state(s). The time course of folding will be detected using a combination of FRET and new environment-sensitive probes. In the cell, protein folding may start at very early stages of peptide synthesis along a narrow tunnel spanning the ribosome, but the characteristic dynamics and conformational changes remain elusive. It is expected that multiparameter single-molecule studies will provide a unique insight into the folding dynamics of nascent peptides, the interaction with the ribosome and with helper biomolecules (chaperones).