Maliwal B P, Kuśba J, Wiczk W, Johnson M L, Lakowicz J R
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201.
Biophys Chem. 1993 May;46(3):273-81. doi: 10.1016/0301-4622(93)80020-j.
We measured distance distributions and end-to-end diffusion coefficients of donor-acceptor pairs linked by a flexible methylene chain using frequency-domain fluorescence energy transfer measurements. The donor was an indole group, and two acceptors with different Förster distances were used. The uncertainties in the recovered parameters describing the end-to-end distance distribution and diffusion coefficient were rather large when each donor-acceptor pair was analyzed separately. It was not possible to resolve distance distributions in the presence of intra-molecular diffusion when the Förster distance was comparable to the mean and half-width of the distribution. Global analysis using two acceptors dramatically improved the resolution. Surprisingly, the Förster distances need not be very different, and a 20% difference between the two R0 values resulted in substantial improvements in resolution. Both the simulations and the experiments suggest the benefit of using global analysis with different Förster distances to obtain reliable distance distribution parameters in the presence of diffusion.
我们使用频域荧光能量转移测量方法，测量了由柔性亚甲基链连接的供体-受体对的距离分布和端到端扩散系数。供体是一个吲哚基团，并使用了两个具有不同Förster距离的受体。当分别分析每个供体-受体对时，描述端到端距离分布和扩散系数的恢复参数的不确定性相当大。当Förster距离与分布的均值和半高宽相当时，在存在分子内扩散的情况下无法解析距离分布。使用两个受体进行全局分析显著提高了分辨率。令人惊讶的是，Förster距离不需要有很大差异，两个R0值之间20%的差异就导致分辨率有显著提高。模拟和实验都表明，在存在扩散的情况下，使用具有不同Förster距离的全局分析来获得可靠的距离分布参数是有益的。
