Quantitative single-molecule conformational distributions: A case study with poly-(L-proline)
Quantitative single-molecule conformational distributions: A case study with poly-(L-proline)
复制标题
DOI：
10.1021/jp055886d
复制
发表时间：
2006-04-20
影响因子：
2.9
通讯作者：
Yang, H
中科院分区：
文献类型：
--
作者：
Watkins, LP;Chang, HY;Yang, H
精确测量平均力的潜力对于基本了解生物大分子的动力学和化学反应性是必要的。最近开发的恒定信息的方法来分析时间依赖性的单分子荧光测量提供的独特优势与最大熵反卷积创建一个程序，用于准确测定分子构象分布，并在这些分布的误差的解析表达式推导。这种新的方法被应用到衍生的聚（L-脯氨酸）系列，P（n）CG（3）K-（生物素）（n = 8，12，15，18，和24），使用模块化的，基于服务器的单三分子光谱仪，能够注册光子到达时间与连续波激发源。为了解释来自微观环境的潜在影响，逐分子校准和校正的因素包括背景、串扰和检测效率。对于每一个单一的聚（L-脯氨酸）分子，尖锐的福斯特型共振能量转移（FRET）效率和距离分布恢复，表明一个静态的端到端的距离上的时间尺度的测量。实验距离进行了比较，不同的刚度模型。结果表明，由高分子量聚L-脯氨酸实验得到的23 A持久长度蠕虫链模型也适用于短链。
Precise measurement of the potential of mean force is necessary for a fundamental understanding of the dynamics and chemical reactivity of a biological macromolecule. The unique advantage provided by the recently developed constant-information approach to analyzing time-dependent single-molecule fluorescence measurements was used with maximum entropy deconvolution to create a procedure for the accurate determination of molecular conformational distributions, and analytical expressions for the errors in these distributions were derived. This new method was applied to a derivatized poly(L-proline) series, P(n)CG(3)K-(biotin) (n = 8, 12, 15, 18, and 24), using a modular, server-based single-triolecule spectrometer that is capable of registering photon arrival times with a continuous-wave excitation Source. To account for potential influence from the microscopic environment, factors that were calibrated and corrected molecule by molecule include background, cross-talk, and detection efficiency. For each single poly(L-proline) molecule, sharply peaked Forster type resonance energy transfer (FRET) efficiency and distance distributions were recovered, indicating a static end-to-end distance on the time scale of measurement. The experimental distances were compared with models of varying rigidity. The results suggest that the 23 A persistence length wormlike chain model derived from experiments with high molecular weight poly(L-proline) is applicable to short chains as well.