Schuler Benjamin, Lipman Everett A, Steinbach Peter J, Kumke Michael, Eaton William A
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, Building 5, Room 104, National Institutes of Health, Bethesda, MD 20892, USA.
Proc Natl Acad Sci U S A. 2005 Feb 22;102(8):2754-9. doi: 10.1073/pnas.0408164102. Epub 2005 Feb 7.
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(0), 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.
为了确定在多肽的单分子荧光实验中，福斯特共振能量转移（FRET）测量是否能够提供定量的距离信息，我们测量了连接到不同长度自由扩散的聚脯氨酸分子末端的供体和受体染料的FRET效率分布。观察到的平均FRET效率与从系综寿命测量确定的值一致，但与将聚脯氨酸视为刚性棒时福斯特理论预期的值有很大差异。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)相当及大于R(0)的距离处，它们则高得多。造成前者的两个可能因素是供体寿命期间不完全的取向平均，以及由于染料尺寸较大，福斯特理论中假设的点偶极近似失效。从朗之万分子动力学模拟获得的端到端距离分布和相关时间表明，较长聚脯氨酸肽的差异可以通过链弯曲来解释，这大大缩短了供体 - 受体距离。
