Polyproline and the "spectroscopic ruler" revisited with single-molecule fluorescence
Polyproline and the "spectroscopic ruler" revisited with single-molecule fluorescence
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DOI：
10.1073/pnas.0408164102
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发表时间：
2005-02-22
影响因子：
11.1
通讯作者：
Eaton, WA
中科院分区：
文献类型：
--
作者：
Schuler, B;Lipman, EA;Eaton, WA
为了确定福斯特共振能量转移（FRET）测量是否可以提供定量的距离信息，在单分子荧光实验的多肽，我们测量的FRET效率分布的供体和受体染料连接到自由扩散的聚脯氨酸分子的末端的各种长度。所观察到的平均FRET效率同意从合奏寿命测量确定的那些，但从福斯特理论预期的值有很大的不同，与聚脯氨酸作为一个刚性棒处理。在供体-受体距离远小于福斯特半径R-0时，观察到的效率低于预测值，而在与R-0相当或大于R-0的距离时，观察到的效率要高得多。两个可能的贡献，前者是不完整的取向平均在捐助者的寿命，因为大尺寸的染料，福斯特理论中假设的点偶极子近似的故障。从Langevin分子动力学模拟获得的端到端的距离分布和相关时间表明，较长的聚脯氨酸肽的差异可以解释为链弯曲，这大大缩短了供体-受体的距离。
To determine whether Forster resonance energy transfer (FRET) measurements can provide quantitative distance information in single-molecule fluorescence experiments on polypeptides, we measured FRET efficiency distributions for donor and acceptor dyes attached to the ends of freely diffusing polyproline molecules of various lengths. The observed mean FRET efficiencies agree with those determined from ensemble lifetime measurements but differ considerably from the values expected from Forster theory, with polyproline treated as a rigid rod. At donor-acceptor distances much less than the Forster radius R-o, the observed efficiencies are lower than predicted, whereas at distances comparable to and greater than R-0, they are much higher. Two possible contributions to the former are incomplete orientational averaging during the donor lifetime and, because of the large size of the dyes, breakdown of the point-dipole approximation assumed in Forster theory. End-to-end distance distributions and correlation times obtained from Langevin molecular dynamics simulations suggest that the differences for the longer polyproline peptides can be explained by chain bending, which considerably shortens the donor-acceptor distances.