Extracting rate coefficients from single-molecule photon trajectories and FRET efficiency histograms for a fast-folding protein.
Extracting rate coefficients from single-molecule photon trajectories and FRET efficiency histograms for a fast-folding protein.
复制标题
DOI：
10.1021/jp1009669
复制
发表时间：
2011-04-28
期刊：
影响因子：
--
通讯作者：
Eaton WA
中科院分区：
文献类型：
--
作者：
Chung HS;Gopich IV;McHale K;Cellmer T;Louis JM;Eaton WA
Recently-developed statistical methods by I.V. Gopich and A. Szabo were used to extract folding and unfolding rate coefficients from single-molecule Förster resonance energy transfer (FRET) data for proteins with kinetics too fast to measure waiting time distributions. Two types of experiments and two different analyses were performed. In one experiment bursts of photons were collected from donor and acceptor fluorophores attached to a 73-residue protein, α3D, freely diffusing through the illuminated volume of a confocal microscope system. In the second, the protein was immobilized by linkage to a surface, and photons were collected until one of the fluorophores bleached. Folding and unfolding rate coefficients and mean FRET efficiencies for the folded and unfolded subpopulations were obtained from a photon by photon analysis of the trajectories using a maximum likelihood method. The ability of the method to describe the data in terms of a two-state model was checked by recoloring the photon trajectories with the extracted parameters and comparing the calculated FRET efficiency histograms with the measured histograms. The sum of the rate coefficients for the two-state model agreed to within 30% with the relaxation rate obtained from the decay of the donor-acceptor cross-correlation function, confirming the high accuracy of the method. Interestingly, apparently reliable rate coefficients could be extracted using the maximum likelihood method, even at low (<10%) population of the minor component where the cross-correlation function was too noisy to obtain any useful information. The rate coefficients and mean FRET efficiencies were also obtained in an approximate procedure by simply fitting the FRET efficiency histograms, calculated by binning the donor and acceptor photons, with a sum of three Gaussian functions. The kinetics are exposed in these histograms by the growth of a FRET efficiency peak at values intermediate between the folded and unfolded peaks as the bin size increases, a phenomenon with similarities to NMR exchange broadening. When comparable populations of unfolded and unfolded molecules are present, this method yields rate coefficients in very good agreement with those obtained with the maximum likelihood method. As a first step toward characterizing transition paths, the Viterbi algorithm was used to locate the most probable transition points in the photon trajectories.
登录
查看更多内容
影响因子：
2.9
作者：
Andrec, M;Levy, RM;Talaga, DS
通讯作者：
Talaga, DS
影响因子：
3.4
作者：
Lee, Tae-Hee;Lapidus, Lisa J.;Chu, Steven
通讯作者：
Chu, Steven
影响因子：
3.4
作者：
Chung, Hoi Sung;Louis, John M.;Eaton, William A.
通讯作者：
Eaton, William A.
影响因子：
3.3
作者：
Gopich, IV;Szabo, A
通讯作者：
Szabo, A
DOI：
10.1073/pnas.0708600104
发表时间：
2007-11-13
影响因子：
11.1
作者：
Hanson, Jeffrey A.;Duderstadt, Karl;Yang, Haw
通讯作者：
Yang, Haw