Lakowicz J R, Kuśba J, Szmacinski H, Gryczynski I, Eis P S, Wiczk W, Johnson M L
Department of Biological Chemistry, School of Medicine, University of Maryland, Baltimore 21201.
Biopolymers. 1991 Oct 15;31(12):1363-78. doi: 10.1002/bip.360311203.
We used time-dependent fluorescence energy transfer, time-dependent collisional quenching, and global analysis of the data resulting from these through-space and contact interactions to recover the end-to-end distance distributions and diffusion coefficients of flexible fluorescent molecules. The fluorescence decays of covalently linked tryptamine-acceptor and tryptamine-quencher pairs were measured by the frequency-domain method. These data were fit using numerical solutions of the differential equation, which predicts the time- and distance-dependent population of the excited state donors in the presence of energy transfer or collisional quenching, followed by transformation to the frequency domain for nonlinear least-squares comparison with the experimental data. We found that the energy transfer data for the donor-acceptor pair alone were adequate to recover the starting distribution and the end-to-end diffusion coefficient; however, the resolution is dramatically improved by the use of both the through-space and contact interactions.
我们使用时间分辨荧光能量转移、时间分辨碰撞猝灭以及对这些通过空间和接触相互作用产生的数据进行全局分析，以恢复柔性荧光分子的端到端距离分布和扩散系数。通过频域法测量共价连接的色胺-受体和色胺-猝灭剂对的荧光衰减。这些数据使用微分方程的数值解进行拟合，该方程预测在存在能量转移或碰撞猝灭的情况下激发态供体的时间和距离依赖性种群，然后转换到频域进行与实验数据的非线性最小二乘比较。我们发现，仅供体-受体对的能量转移数据就足以恢复起始分布和端到端扩散系数；然而，通过同时使用空间相互作用和接触相互作用，分辨率得到了显著提高。
