Investigations of energy trapping in photosynthesis and of DNA looping by endonuclease.

摘要

Fluorescence resonance energy transfer (FRET) was used to study energy transfer processes in photosynthetic pigment-protein complexes and to investigate protein-DNA interactions at the single molecule level. Energy transfer in chromatophores isolated from strains of the purple bacterium Rhodobacter sphaeroides containing wild-type (WT) Light Harvesting Complex I (LHI) or mutant LHI (mutation tryptophan at position 43 to phenylalanine) and either a WT Reaction Center (RC) or a RC with mutations that result in a P/P+ midpoint potential either above or below that of WT RCs was studied at temperatures between 294 K and 10 K. In the RC mutants with the highest P/P+ midpoint potentials, the initial electron transfer rate decreased significantly, and at low temperatures it was possible to directly observe energy transfer from LHI to the RC by detecting the fluorescence kinetics from both protein complexes. A unique model describing fluorescence kinetics from different samples was developed to explain multi-exponential fluorescence decays. The energy transfer from the LHI mutant (for which absorption and fluorescence spectra were blue-shifted by 21 nm compared to WT LHI) to the RCs occurred about two times faster compared to energy transfer from WT LHI to RCs. The 2-fold acceleration of energy transfer in the LHI mutant was consistent with the increase in the energy transfer rate estimated from the spectral overlap between the LHI fluorescence and RC absorbance according to Forster energy transfer theory.; DNA looping dynamics were investigated at the single molecule level using FRET occurring between Cy3 and Cy5 dyes on the ends of a 172 base-pair long DNA molecule containing two NgoMIV restriction sites. DNA/protein complex formation at the single molecule level was observed in the presence of the NgoMIV restriction endonuclease. The FRET efficiency distributions revealed a population of complexes exhibiting energy transfer efficiency of 30%. This corresponded to a distance about 60 A between DNA ends, which correlated well with the crystal structure of Ngo MIV. Dynamics of DNA loop formation were assessed by studying DNA-protein interactions using immobilized DNA molecules. DNA loop formation occurred with a rate constant of about 12.5 s-1, while the looped DNA-protein complex dissociated with a rate constant of about 15.5 s-1.