AvGFP Protein

The protein matrix of AvGFP efficiently forbids significant torsional motions of the chromophore, leading to near-maximum and highly homogeneous green fluorescence emission (see Sect. 3.1). Failure to do so results in weakly or non-fluorescent GFPs [113-115], while it was shown recently that the differences in... 

Ultrafast ESPT from the neutral form readily explains why excitation into the A and B bands of AvGFP leads to a similar green anionic fluorescence emission [84], Simplistic thermodynamic analysis, by way of the Forster cycle, indicates that the excited state protonation pK.J of the chromophore is lowered by about 9 units as compared to its ground state. However, because the green anionic emission is slightly different when it arises from excitation into band A or band B (Fig. 5) and because these differences are even more pronounced at low temperatures [81, 118], fluorescence after excitation of the neutral A state must occur from an intermediate anionic form I not exactly equivalent to B. State I is usually viewed as an excited anionic chromophore surrounded by an unrelaxed, neutral-like protein conformation. The kinetic and thermodynamic system formed by the respective ground and excited states of A, B, and I is sometimes called the three state model (Fig. 7). 

The chromophore of ECFP does not bear the deprotonable phenol that is crucial to the photophysics of most AvGFP variants, and displays a markedly different spectroscopy. It has been quickly recognized that, despite its prominent interest in biological applications, the properties of this variant are suboptimal. Indeed, while the brightness of the protein is relatively low (eM = 32,000 M 1 cm-1, fluorescence emission is both spectrally and kinetically heterogeneous. The fluorescence comprises two major decay times at 3.6 ns and 1.3 ns... 

The crystal structure of avGFP was first solved in 1996, independently by two groups [23, 24], The structure revealed a cylindrical protein, consisting of 11 /1-strands (Fig. 5.1), which was named 11-stranded /1-barrel. A single a-helix runs along the axis inside the /1-barrel and was found to contain the chromophore, the source... 

Cyan fluorescent proteins (CFPs) have blue-shifted excitation and emission spectra, because of the mutation Tyr66Trp inside the chromophore (Fig. 5.3C) [34], CFP fluorescence (Ex 435 nm/Em 474 nm) is less blue-shifted than for EBFP and CFP excitation is intermediate to the excitation of the neutral and anionic chromo-phores of avGFP [4], CFPs are widely used for dual-color imaging and FRET applications together with yellow fluorescent proteins (YFP, Section 3.6). 

With green fluorescent proteins becoming available from other organisms scientists started to add the initial letters of the latin names of the organisms as a prefix to the name of the gene. However, this system is not used consequently. Hence, in the literature the wildtype - GFP is also known as AvGFP, but even nowadays in the majority of publications only the term GFP is used for the GFP 1-gene form Aequorea victoria. 

Until recently, the green fluorescent protein (avGFP) from the jellyfish Aequorea victoria was the only fluorescent protein to be widely mutated and altered for biological apphcations. Meanwhile, a family of GFP-like proteins from different organisms has emerged that share the common fl-barrel fold structure and intrinsic chromophores but represent a vast spectral range, with (E)GFP and DsRed being the most prominent representatives [33]. 

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