The recovery of dipolar relaxation times from fluorescence decays as a tool to probe local dynamics in single tryptophan proteins.
The dipolar relaxation process induced by the excitation of the single tryptophan residue of four proteins (staphylococcal nuclease, ribonuclease-T1, phosphofructokinase, and superoxide dismutase) has been studied by dynamic fluorescence measurements. A new algorithm taking into account the relaxation effect has been applied to the fluorescence decay function obtained by phase-shift and demodulation data. This approach only requires that fluorescence be collected through the whole emission spectrum, avoiding the time-consuming determination of the data at different emission wavelengths, as usual with time-resolved emission spectroscopy. The results nicely match those reported in the literature for staphylococcal nuclease and ribonuclease-T1, demonstrating the validity of the model. Furthermore, this new methodology provides an alternative explanation for the complex decay of phosphofructokinase and human superoxide dismutase suggesting the presence of a relaxation process even in proteins that lack a lifetime-dependent spectral shift. These findings may have important implications on the analysis of small-scale protein dynamics, since dielectric relaxation directly probes a local structural change around the excited state of tryptophan.
Mei
Giampiero
G
Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Rome 00133, Italy.
Di Venere
Almerinda
A
De Matteis
Fabio
F
Rosato
Nicola
N
D000465:Algorithms; D004798:Enzymes / Q000737:chemistry*; D008836:Micrococcal Nuclease / Q000737:chemistry; D008958:Models, Molecular; D009038:Motion; D025221:Phosphofructokinases / Q000737:chemistry; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D006163:Ribonuclease T1 / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013482:Superoxide Dismutase / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry*
D004798:Enzymes; D011506:Proteins; D014364:Tryptophan; D013482:Superoxide Dismutase; D025221:Phosphofructokinases; D006163:Ribonuclease T1; D008836:Micrococcal Nuclease