Quenchers induce wavelength dependence on protein fluorescence lifetimes.
We have analysed the picosecond resolved fluorescence emission decay of horseradish peroxidase A2 and of HEW lysozyme acquired with a streak camera. Analyses of the fluorescence decay data of both proteins revealed that the dynamics of the decay is dependent on the emission wavelength. Our data strongly indicates that resonance energy transfer occurring between aromatic residues and different protein fluorescence quencher groups, and the nature of the quencher groups, are the causes of the observed wavelength dependent mean lifetime distribution. Using the global analysis data to calculate the fluorescence mean lifetime at each wavelength revealed that for lysozyme, the mean fluorescence lifetime increased with observation wavelength, whereas the opposite was the case for peroxidase. Both proteins contain strong fluorescence quencher groups located in close spatial proximity to the protein's aromatic residues. Lysozyme contains disulfide bridges as the main fluorescence quencher whereas peroxidase contains a heme group. Both for lysozyme and horseradish peroxidase there is a clear correlation between the observed fluorescence mean lifetime of the protein at a particular emission wavelength and the respective quencher's extinction coefficient at the respective wavelength. Furthermore, our study also reports a comparison of the analyses of the fluorescence data done with three different methods. Analyses of the fluorescence decay at 10 different fluorescence emission wavelengths revealed significant differences in both fluorescence lifetimes and the pre-exponential factor distributions. Such values differed from the values recovered from the integrated decay curves and from global analyse.
Klitgaard
Søren
S
NanoBiotechnology Group, Department of Physics and Nanotechnology, Aalborg University, Skjernvej 4C, Aalborg, Denmark.
Neves-Petersen
M T
MT
Petersen
S B
SB
D000085:Acetates / Q000737:chemistry; D002021:Buffers; D002951:Citrates / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D005453:Fluorescence; D006418:Heme / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000528:radiation effects*; D006863:Hydrogen-Ion Concentration; D009113:Muramidase / Q000737:chemistry* / Q000528:radiation effects*; D012996:Solutions / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014325:Tromethamine / Q000737:chemistry
D000085:Acetates; D002021:Buffers; D002951:Citrates; D004220:Disulfides; D012996:Solutions; D014325:Tromethamine; D006418:Heme; D006735:Horseradish Peroxidase; D009113:Muramidase