One-Photon and Two-Photon Excitation of Fluorescent Proteins - Fluorescent Proteins: From Understanding to Design - page 26

(Table 1 ). When the X-ray structure is not available, the assignment of the chromo-
phore structure may be doubtful. The chromophore of mOrange and mBanana
contains a structural modification at the acylimine tail; so the blueshifted optical
properties of these FPs cannot be wholly attributed to chromophore-protein non-
covalent interactions. As such, the original DsRed is among the most blueshifted
FPs with the DsRed chromophore structure (Table 6 ).
Mutation K83M was found to redshift the optical properties by dislocating
Lys70, which in wild-type DsRed is in close contact with the chromophore-bridg-
ing carbon (at 3.6 ˚ distance) [ 45 , 103 ]. A similar mechanism is at play in mCherry
and mStrawberry, both featuring the analogous K83L mutation. Further redshifting
in these two mutants is provided by the H-bond between protonated E215 and the
nitrogen in the chromophore imidazolinone (see Fig. 9 ). It is indeed observed that at
basic pH conditions ( p Ka ~10), at which this H-bond is presumably removed due to
deprotonation of E215, the absorption peak of mCherry and mStrawberry blueshifts
from 584 to 566 nm and from 574 to 548 nm, respectively. A third source of
redshifting is achieved in mCherry/mStrawberry by the additional K163Q/K163M
mutations eliminating one H-bond to the chromophore phenolate.
Like in yellow mutants of av GFP,

-orbital stacking of the chromophore with a
Tyr residue results in a redshift. For instance, the protein mGrape3 with the I197Y
mutation (corresponding to av 203) is remarkably redshifted (608 ex and 646 em).

p

Fig. 9 Relevant residues and water molecules in the chromophore environment of red (DsRed and
mCherry) and far-red (mPlum and mNeptune) FPs. The coordinates were taken from the Protein
Data Bank, with codes 1ZQO [ 105 ], 2H5Q [ 45 ], 2QLG [ 106 ], and 3IP2 [ 41 ]. The DsRed structure
contains both mature and immature chromophore conformations. Only the mature chromophore is
shown here. The Arginine residue at position 95 is omitted for clarity