Combining MFD and PIE for Accurate Single-Pair Forster Resonance Energy Transfer Measurements

被引:142
作者
Kudryavtsev, Volodymyr [1 ,2 ]
Sikor, Martin [1 ,2 ]
Kalinin, Stanislav [3 ]
Mokranjac, Dejana [4 ]
Seidel, Claus A. M. [3 ]
Lamb, Don C. [1 ,2 ,5 ]
机构
[1] Univ Munich, Dept Chem, Munich Ctr Integrated Prot Sci CiPSM, D-81377 Munich, Germany
[2] Univ Munich, Ctr Nanosci, D-81377 Munich, Germany
[3] Univ Dusseldorf, Inst Phys Chem 2, D-40225 Dusseldorf, Germany
[4] LMU Munchen, Inst Physiol Chem, D-81377 Munich, Germany
[5] Univ Illinois, Dept Phys, Urbana, IL 61801 USA
关键词
DnaK; FRET; multiparameter fluorescence detection; pulsed interleaved excitation; single-molecule studies; FLUORESCENCE CORRELATION SPECTROSCOPY; PROBABILITY-DISTRIBUTION ANALYSIS; MOLECULE FLUORESCENCE; STRUCTURAL HETEROGENEITIES; CONFORMATIONAL TRANSITIONS; PHOTON DISTRIBUTION; LIFETIME ANALYSIS; AQUEOUS-SOLUTION; HSP70; CHAPERONE; FRET;
D O I
10.1002/cphc.201100822
中图分类号
O64 [物理化学(理论化学)、化学物理学];
学科分类号
070304 ; 081704 ;
摘要
Single-pair Forster resonance energy transfer (spFRET) experiments using single-molecule burst analysis on a confocal microscope are an ideal tool to measure inter- and intramolecular distances and dynamics on the nanoscale. Different techniques have been developed to maximize the amount of information available in spFRET burst analysis experiments. Multiparameter fluorescence detection (MFD) is used to monitor a variety of fluorescence parameters simultaneously and pulsed interleaved excitation (PIE) employs direct excitation of the acceptor to probe its presence and photoactivity. To calculate accurate FRET efficiencies from spFRET experiments with MFD or PIE, several calibration measurements are usually required. Herein, we demonstrate that by combining MFD with PIE information regarding all calibration factors as well as an accurate determination of spFRET histograms can be performed in a single measurement. In addition, the quality of overlap of the different detection volumes as well as the detection of acceptor photophysics can be investigated with MFD-PIE. Bursts containing acceptor photobleaching can be identified and excluded from further investigation while bursts that contain FRET dynamics are unaffected by this analysis. We have employed MFD-PIE to accurately analyze the effects of nucleotides and substrate on the interdomain separation in DnaK, the major bacterial heat shock protein 70 (Hsp70). The interdomain distance increases from 47 angstrom in the ATP-bound state to 84 angstrom in the ADP-bound state and slightly contracts to 77 angstrom when a substrate is bound. This is in contrast to what was observed for the mitochondrial member of the Hsp70s, Ssc1, supporting the notion of evolutionary specialization of Hsp70s for different cellular functions in different organisms and cell organelles.
引用
收藏
页码:1060 / 1078
页数:19
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