Engineering single-molecule fluorescence dynamics for advanced biomolecular applications

The green fluorescent protein (GFP) of the Aequorea victoria jellyfish is a uniquely fluorescent label that is opening the way to advanced biomolecular studies. Fundamental processes on a nanoscopic scale can, in fact, be directly addressed by imaging and spectroscopy of GFP-tagged molecules. To this end, the design of labels with novel spectral and dynamic properties plays an important role to enhance GFP applicability. We investigated and engineered the fluorescence dynamics of two GFP mutants, the F64L/S65T and the F64L/S65T/T203Y, down to the single-molecule level in order to evaluate their suitability for a broad range of experimental systems. We shall argue that the first mutant is suitable for quantitative fluorescence microscopy and displays fluorescence almost unaffected by the environment, while the second offers the important additional advantage of undergoing photobleaching reversal after ultraviolet illumination. As a consequence, the latter mutant is a viable candidate for applications when prolonged imaging is required. As an example of novel possibilities provided by GFP technology, we shall show studies of HIV-1 Tat activity in cells and, in particular, we shall demonstrate Tat interaction with cyclin T1 by fluorescence-resonance energy transfer.