One-Photon and Two-Photon Excitation of Fluorescent Proteins - Fluorescent Proteins: From Understanding to Design - page 27

It is, however, characterized by low quantum yield (0.03) and complex photophy-
sics possibly connected with cis - trans isomerization of the chromophore [ 41 ].
As the

-conjugated system in the RFP chromophore reaches down to the
acylimine group, variations in the surrounding environment can also modify the
optical properties. For example, mPlum, one very redshifted DsRed variant, features
(besides the redshifting K163M mutation and the E215-imidazolinone H-bond) a
H-bond between (presumably neutral) Glu16 and the acylimine oxygen. This
H-bond is absent in DsRed and most other mutants, where position 16 is occupied
by hydrophobic amino acids. It is found that mutation of Glu16 strongly affects the
emission peak while leaving the excitation almost unchanged. For example,
replacing Glu16 with Leu blueshifts the emission peak from 649 to 630 nm,
while the excitation peak is only slightly modified from 590 to 588 nm [ 106 ].
Likewise, in mNeptune [ 41 ], a far-red FP derived from eqFP578 of Entacmaea
quadricolor (not to be confused with eqFP611) [ 107 ], the acylimine oxygen is
H-bonded to a water molecule. The slightly blueshifted optical properties of mKate
(another eqFP578 mutant) can be attributed to the absence of this H-bond, caused
by Met41 displacing the hydrogen-bonded water molecule. As is the case with
many FPs, the red shift of mNeptune is, however, the result of multiple entangled
contributions. As a consequence, it may be difficult to predict the outcome of a
mutation. Quite surprisingly, for example, introducing the Tyr at 197 in mNeptune
(R197Y mutant), at the presumably

p

-stacked position with the phenolate, blue-
shifts, rather than redshifts, the optical response [ 41 ].
Chromophore deviation from coplanarity is yet another determinant of optical
properties. In general, distorted chromophore structures are associated with
poor quantum yield of fluorescence. For instance, mCherry is characterized by a
particularly distorted chromophore and a rather low fluorescence quantum yield
(0.22) [ 7 ]. Chromophore deviation from coplanarity can be quantified by the twist
around

p

(see Table 2 ), or also including the tilt angle defined as the deviation
of the phenol oxygen from the perfectly planar chromophore [ 9 ].

t and f

6 Two-Photon Excitation

Despite their relevance in multiphoton fluorescence microscopy applications, two-
photon spectroscopic features of FPs have been somewhat neglected in the past years,
appearing in few experimental works centered on BFP, CFP, EGFP, and DsRed
[ 108 - 110 ]. More recently, parallel but independent experimental [ 67 , 111 , 112 ]and
theoretical [ 113 ] investigations extended such analyses to several other FPs.
Figures 10 and 11 report the two-photon excitation spectra of, respectively, blue/
cyan/green and orange/red FPs. One-photon spectra, suitably scaled in the abscissae,
are also reported for comparison. In all investigated proteins, the long-wavelength
band (i.e., the main excitation band) is active also at two-photon excitation,
implying that the first singlet excitation is characterized by both one and two-
photon oscillator strength. This statement is verified for all chromophores examined