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Arch Biochem Biophys. 2003 Sep 15;417(2):159-64.

The recovery of dipolar relaxation times from fluorescence decays as a tool to probe local dynamics in single tryptophan proteins.

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Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Rome 00133, Italy.


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.

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