Fluorescence spectroscopy

Abstract

Although providing extensive detail, the time-resolved experiments described here can be quite complex and the instrumentation is not always readily available. However, a number of national fluorescence user facilities funded by the National Institutes of Health or the National Science Foundation are currently in operation and are dedicated to the use of fluorescence spectroscopy in the biomedical and biophysical sciences. These centers include the Laboratory for Fluorescence Dynamics at the University of Illinois and the Center for Fluorescence Spectroscopy at the University of Maryland. Even in the absence of the sophisticated equipment necessary for carrying out the time-resolved experiments, a great deal of information can be obtained from steady-state fluorescence profiles if one is careful to monitor all of the available fluorescence observables, namely intensity, wavelength or color, and polarization. Steady-state measurements of ANS binding are also quite informative. The combination of kinetic as well as equilibrium approaches, as with folding studies using any technique, will provide further insight into the pathways and stable and transient intermediates in the folding and unfolding reactions. Fluorescence spectroscopy offers a very sensitive window into the structural and dynamic characteristics of macromolecules. Recent advances in data acquisition and analysis combined with available structure information from NMR and crystallographic studies have led to increasingly greater insight into the structural and dynamic determinants of fluorescence decay parameters in the native states of proteins. As our understanding of the fluorescence properties of native proteins has grown, fluorescence spectroscopists have begun to investigate what fluorescence can tell us about the denatured states of proteins as well as the folding/unfolding transitions and pathways. A great deal of progress has been made in the characterization and interpretation of the response of the various fluorescence parameters to protein folding and denaturation. There remain, however, a number of unanswered questions, particularly concerning the structural and dynamic determinants of the fluorescence properties of the denatured states of proteins. Future studies will undoubtedly be aimed toward this goal, and progress in this area will certainly result from systematic comparisons of fluorescence studies with a number of other biophysical and biochemical approaches.