One-Photon and Two-Photon Excitation of Fluorescent Proteins - Fluorescent Proteins: From Understanding to Design - page 2

5.1 Proteins with GFP Chromophore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
5.2 Proteins with RFP Chromophore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
6 Two-Photon Excitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
7 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

1 Introduction

By the term fluorescent proteins (FPs), it is customary to indicate all fluorescent
homologues of the original Aequorea victoria green fluorescent protein (GFP or
av GFP). An accessory protein of the bioluminescence system of jellyfish A. victo-
ria , av GFP was discovered as early as the 1960s [ 1 ]. Thirty years later, with the
cloning of the gene [ 2 ] and the demonstration that its expression in other organisms
generates fluorescence [ 3 , 4 ], interest in GFP began to rise dramatically. Since then,
it has triggered a revolution in bioimaging by fluorescence microscopy [ 5 ]. Soon,
many other fluorescent and nonfluorescent GFP homologues were discovered in a
variety of sea organisms, such as reef corals and sea anemones [ 6 ]. Further
discoveries and mutagenesis engineering have produced a profusion of FPs with
optical properties spanning most of the visible spectrum and beyond.
Fluorescence in FPs stems from the presence of a chromophore moiety, formed
within the conserved b -barrel fold via a mechanism entailing autocatalytic back-
bone cyclization at an internal tripeptide sequence. Distinct postcyclization proces-
sing leads to different chromophore structures. The multiplicity of optical
properties of FPs is surely one of the factors that contribute to their usefulness. It
primarily arises from the different chemical structures of the chromophore. A finer
tuning originates from the noncovalent interactions of the chromophores with the
surrounding molecular matrix.
This chapter focuses on the mechanisms behind this spectral tuning, covering
both experimental and theoretical/computational work. The reader is first presented
with the more familiar case of av GFP. The chromophore structures of other FPs are
described in Sect. 3 . The following section surveys various studies on synthetic
analogs of chromophores of FPs in gas phase and in solution. Section 5 provides a
detailed description of spectral modifications due to interactions between chromo-
phore and surrounding protein matrix. Finally, the last section covers two-photon
properties of FPs. Several other reviews on FP optical properties are available,
some treating more exhaustively the variety of GFP-like fluoro and chromoproteins,
and other more focused on application purposes. For recent surveys, see [ 7 - 9 ].

2 av GFP: Structure and Optical Properties

The first to be cloned [ 2 ] and functionally expressed in other organisms [ 3 , 4 ],
av GFP has actually been replaced in most applications by its mutants and homo-
logues. Nonetheless, being one of the best characterized in terms of optical and
photophysical properties, it is a suitable starting point to introduce the concepts
recurring in this chapter.