A PPS'99 Project

The Use of Fluorescence in Research into Protein Structures

by Gabor Mocz

These pages are directed to illustrate some aspects of fluorescence spectroscopy that are particularly useful for elucidating structural and dynamic properties of proteins. Fluorescent techniques can provide valuable parameters consistent with crystallographic results. The various environments of the fluorophores of a folded protein and the unique stereochemistry of the polypeptide chain affect the fluorophores in many ways which can be used to characterize and to follow changes in the folded conformation in solution. Fluorescence-based techniques can give information about a protein's conformation, binding sites, solvent interactions, degree of flexibility, internal motions, rotational diffusion coefficient, and many other physicochemical parameters.

Topics covered in these pages include:

Basic Definitions and Phenomena of Fluorescence

Intrinsic Fluorescence of Proteins and Peptides

The Fluorophore of Green Fluorescent Protein

The Local Environment of Tryptophan in Melittin

Quenching of Fluorescence to Measure Accesibility of Tryptophan Residues

Fluorescent Resonance Energy Transfer as a Probe of Proximity in Proteins

Fluorescence Anisotropy and Polarization to Detect Internal Protein Motions

Dynamics of Protein Folding

The text is not intended as a comprehensive review of the current (huge and overhelming!) literature. Instead, it stresses the underlying principles and gives some examples from a few selected areas. Instrumentation and other related areas such as confocal laser scanning microscopy and two-photon molecular excitation microscopy for three-dimensional measurements of biological structures will not be discussed in these pages. You can find more information in:

References

gmocz@hawaii.edu