One-Photon and Two-Photon Excitation of Fluorescent Proteins - Fluorescent Proteins: From Understanding to Design - page 21

alignment. When no such prefix is present, the number refers to the normal position
within the sequence.
5.1.1 Cyan/Teal Group
The cyan/teal group contains FPs isolated from reef corals, such as Discosoma
striata (dsFP483), Anemonia majano (amFP486), and Clavularia (cFP484), as well
as two cFP484 mutants, mTFP0.7 and mTFP1. These cyan-emitting proteins are
naturally tightly bound tetramers; hence, av GFP mutants bearing a Trp at av 66 are
more popular as cyan-emitting FPs, their cyan fluorescence being due to a different
chromophore structure (CFP in Fig. 4 ). Recently, however, the mTFP mutants were
engineered to avoid oligomerization and exist as monomers [ 89 ]. Understanding
the origin of the blueshifted spectral properties of this group has proven rather
challenging. The broad absorption/excitation band hints at structural heterogeneity
of either the chromophore or the surroundings [ 86 ]. By contrast, the narrower
emission spectrum signals a unique emitting state.
AmFP486, cFP484, and the mTFPs (but not dsFP483) feature a quadrupole
network of salt bridges beneath the chromophore, involving one Arg (at av 69),
one His (at av 203), and two Glu (at av 150 and av 222). The His, being hydrogen
bonded on both sides to the Glu, is presumably cationic [ 87 ]. In amFP486, substitu-
tion of this His with Thr resulted in redshifted excitation and emission (from 454 to
470 nm in absorption and from 486 to 515 nm in emission), accompanied by a
decline in fluorescence quantum yield and heterogeneity of chromophore structure
[ 87 ]. In mTFP1, a major role is played by His163 ( av 165). Mutation of His163 into
Met abolishes its H-bond with the chromophore phenolate and redshifts the excita-
tion/emission maximum (from 462/492 to 487/503 nm) [ 89 , 91 ]. Introducing the
further T73A mutation additionally shifts the excitation/emission to 498/515 nm,
possibly by perturbing the quadrupole arrangement supporting the cationic His197
[ 91 ]. The quadrupole arrangement is, however, neither sufficient nor necessary for
cyan emission. It is present in Dronpa, a green -emitting FP, and it is missing in cyan
dsFP483, in which His at av 203 is replaced by a Thr. However, in dsFP483, a
different charged residue, a lysine (K70), is in close contact with the chromo-
phore-bridging carbon and points toward the phenolate [ 86 ], suggesting that such
proximity of a charged residue is indeed necessary.
Inspection of the various chromophore-environment configurations in the rele-
vant X-ray structures (reported in Fig. 7 ) highlights another important - and
possibly necessary - feature common to these cyan-emitting proteins, namely the
presence and arrangement of three H-bond donors facing the chromophore pheno-
late. Indeed, a water molecule and Ser at position av 148 are present in all examined
structures and at H-bonding distance to the phenolate. The third H-bond is provided
either by His at av 167 (in dsFP483, cFP484, and in the mTFPs), or by a water
molecule in amFP486, occupying a cavity set empty by the small bulk of Ala at
av 167. Neither Dronpa nor cmFP512 (both belonging to the green group), though
featuring the quadrupole arrangement, has all three H-bond donors: His av 167 is