Fluorescence Spectroscopy

  • Catherine A. Royer
Part of the Methods in Molecular Biology™ book series (MIMB, volume 40)


The intrinsic fluorescence of aromatic amino acids in proteins has long been used as a means of monitoring unfolding/refolding transitions induced by chemical denaturants, temperature, pH changes, and pressure. The fluorescence properties of tryptophan residues in particular are exquisitely sensitive to perturbations of protein structure, whereas the low quantum yields of phenylalanine and tyrosine render these probes somewhat less useful for such studies. Accordingly, the following discussion of the application of fluorescence techniques to the study of protein folding will be limited to the intrinsic fluorescence of tryptophan in proteins and a few examples of the use of extrinsic fluorescent dyes. Although changes in fluorescence properties have been widely used to construct equilibrium and kinetic protein folding and unfolding profiles, rarely is there a detailed physical interpretation of these changes. Generally, protein folding is well described by a two-state process, and the profiles obtained monitoring fluorescence are coincident with those obtained by circular dichroism. In these cases, the fluorescence profiles are taken as indicators of general loss of protein structure, even though they arise from changes that are local to the tryptophan residue(s). In a number of studies, however, the fluorescence unfolding/refolding profiles, because they arise from structural changes local to particular tryptophan residues, have revealed non-two-state behavior (1, 2, 3). Thus, in some cases, fluorescence profiles can yield information about transient and stable intermediates in the folding/unfolding transition.


Tryptophan Residue Rotational Correlation Time Denature State Folding Intermediate Denaturant Concentration 
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Copyright information

© Humana Press Inc. 1995

Authors and Affiliations

  • Catherine A. Royer
    • 1
  1. 1.School of PharmacyUniversity of WisconsinMadisonWI

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