Fig. 11 19 F NMR analysis of ECFP[(6-F)Trp] and modeling of the chromophore's vicinity.
19 F NMR upon fluorotryptophan incorporation into ECFP revealed a set of four signals for two
residues [Trp57 and Trp66, the spectrum of EGFP[(6-F)Trp] is given as example (a)]. Therefore, it
is reasonable to assume that the fluorinated Trp residues exist in two states. As flipping of the
aromatic residues and phenomena such as aggregation can be excluded as reason for this observa-
tion (see text for a detailed discussion) modeling of the chromophore's vicinity was performed (b).
Thereby, it turned out that Tyr145 and His148 might be able to adopt two conformations resulting
in a different electronic environment of the Trp66 residue responsible for the splitting of the NMR
signal. For Trp57, a similar explanation may holds true
His148 is directed toward the solvent. Both residues are involved in interactions
with the chromophore. Conversely, in the “minor” conformation Tyr145 is turned
toward the solvent without any interaction with the chromophore and His148 is
directed toward the protein. At this point, it is worth to note that in EGFP both
residues are directed to the chromophore [ 11 ]. Moreover, the two different confor-
mational states could only be detected in solution ( 19 FNMR). In the crystal structure
of ECFP[(4-F)Trp], it was not possible to detect two conformations [ 15 ].
However, pH titration does not affect the emission profile of the protein as
would be expected by a changed His148 conformation. Most probably, hydropho-
bic residues in the neighborhood hinder the entry of water molecules or the
imino nitrogen is involved in a hydrogen bond and therefore not accessible for
titration [ 58 ]. In addition, the observed enthalpy-entropy compensation is in agree-
ment with a flipping His148 [ 58 ]. At this point, it is worth to note that the two
conformational states are not the reason for the complex photophysics of ECFP
( vide supra ).
Interestingly, the crystal structure of ECFP[(4-F)Trp] reveals an interaction
between the fluorine atom and the sulfur atom of M218 [ 15 ]. Sulfur is highly
polarizable and is known to interact with aromatic residues [ 67 , 68 ]. The preferred
distance between sulfur and the ring centroid is r
6 ˚ and in the case of ECFP
[(4-F)Trp] fluorine and sulfur posses a distance of 3.5 ˚ . According to the known
interaction geometries, the sulfur points also toward one of the ring hydrogen of
(4-F)Trp66 [ 15 ].
In contrast to the fluorine analogs, incorporation of 4-aminotryptophan ((4-Am)
Trp) seems not to induce two conformational states of the chromophore in ECFP