Single-molecule optical spectroscopy of autofluorescent proteins

Abstract

Several genetically encoded autofluorescent proteins are now available, in which the emission arises from the formation of a fluorophore directly from the amino acids of the protein sequence. These proteins are heavily used in cellular biology to assess gene expression and protein localization. The optical behavior of such proteins has recently been described in vitro at the single-molecule level. These investigations have explored several yellow-emitting mutants of the green fluorescent protein (GFP), the red-emitting DsRed protein, and the dual-GFP cameleon construct designed for fluorescence resonant energy transfer sensing of calcium ion binding. A variety of blinking, switching, and fluctuation effects have been observed, showing that several dark states are accessible after optical excitation, arising from variations in charge state, isomerization state, and photobleaching. The single-molecule spectroscopy of these systems will be surveyed by describing several illustrative examples. Although the autofluorescent proteins available at present need improvement in order to reach the performance of laser dye fluorophores, the novelty of these systems and their ease of use in biological contexts requires a continuing effort to understand the photophysical behavior.