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

Arch Biochem Biophys. 2003 Sep 15;417(2):159-64. doi: 10.1016/s0003-9861(03)00351-5.

Abstract

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.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Algorithms*
  • Enzymes / chemistry*
  • Micrococcal Nuclease / chemistry
  • Models, Molecular
  • Motion
  • Phosphofructokinases / chemistry
  • Protein Conformation
  • Proteins / chemistry*
  • Ribonuclease T1 / chemistry
  • Spectrometry, Fluorescence / methods*
  • Superoxide Dismutase / chemistry
  • Tryptophan / chemistry*

Substances

  • Enzymes
  • Proteins
  • Tryptophan
  • Superoxide Dismutase
  • Phosphofructokinases
  • Ribonuclease T1
  • Micrococcal Nuclease