Primary Photophysical Processes in Chromoproteins - Fluorescent Proteins: From Understanding to Design - page 56

overall rate of proton transfer. Such details are yet to be incorporated into theoreti-
cal calculations, but when they are a picture of long range proton transfer in proteins
may emerge with significance beyond that of understanding ESPT in avGFP.
5 Photochemistry and Photochromism
Photochemistry may induce a permanent change in the absorption spectrum of a
chromophore under irradiation. In some cases, such a photochemical change is
reversible, either thermally on a ground state potential surface or photochemically
through excitation of the product state, in which case the phenomenon is called
photochromism [ 121 ]. Photochromism has been known for many years in organic
photochemistry where the colour change, and the structure change which often
accompanies it, have a number of important applications [ 121 ]. However, the
observation of photochemistry and photochromism in CPs has been a source of
immense excitement and intense research activity [ 49 , 122 - 125 ]. That early excite-
ment has been more than justified by the important applications which have been
demonstrated. For example, the ability to induce photochemically a permanent
change in the colour of a spatially localised population of labelled proteins (optical
highlighting [ 28 , 29 ]) permits the tracking of selected sub-cellular structures. The
applications of CP photochromism have proven even more remarkable. The ability
to switch a protein between bright and dark states at will forms the basis of a novel
method of super-resolution optical microscopy. Typically, resolution in an optical
microscope is limited by the Rayleigh criterion to be no better than 200 nm [ 31 , 32 ].
The position of a single fluorescent molecule may, however, be located within a few
tens of nanometers by straightforward fitting procedures, provided there are no
interfering molecules close by. However, there is no improvement in resolution if
the fluorophores must be kept so far apart. The advantage of photoswitchable or
kindling protein labels is that a dense population of fluorophores may be switched
between dark and bright states, which permits the accumulation of a series of
isolated fluorophore images with tens of nanometre resolution which, when com-
bined, give a complete image with ultrahigh resolution [ 30 - 32 ].
A comprehensive description of photoconversion in CPs has been presented by
van Thor elsewhere in this volume. In this section, we focus only on the primary
photophysics, and specifically on how the photoconversion mechanism may relate
to the phenomena of proton transfer and ultrafast IC described above.
Miyawaki and co-workers reported the remarkable observation that a protein
isolated from coral possessing a chromophore with structure and spectroscopy
similar to avGFP underwent photoconversion to create a red light-absorbing and -
emitting species upon irradiation in the blue region of the spectrum [ 27 ]. The
protein was named kaede (maple leaf in Japanese) which elegantly suggests its
behaviour. Shortly later, very similar observations were made in the protein EosFP
also isolated from coral [ 124 ]. Structural studies showed that the chemical struc-
ture of the red-emitting chromophore differs markedly from that of the GFP