Fluorescent Protein-Based Biosensors and Their Clinical Applications - Progress in Molecular Biology and Translational Science - page 316

Table 8.1 Amino acid sequences of the chromophores of fluorescent proteins
Amino acid position

64 65 66 67 68 69

Aequorea GFP Phe Ser Tyr Gly Val Gln

GFP-S65T Phe Thr Tyr Gly Val Gln

EGFP Leu Thr Tyr Gly Val Gln

EBFP Leu Thr His Gly Val Gln

ECFP Leu Thr Trp Gly Val Gln

EYFP Phe Gly Tyr Gly Val Gln

DsRed Phe Gln Tyr Gly Ser Lys

(65) (66) (67) (68) (69) (70)

The actual amino acid numbers for DsRed are shown in parentheses. Amino acids at positions of 65-67
are directly involved in chromophore formation.

protein). Despite attempts to improve the photochemical characteristics of
these blue- or cyan-emitting GFP homologs, blue-emitting fluorescent pro-
teins (possessing histidine at position 66) exhibit low brightness in compar-
ison with their green- or cyan-emitting counterparts. Recently, Dr. Nagai
and colleagues succeeded in developing a bright ultramarine fluorescent
protein, Sirius, by introducing Trp66Phe and several additional mutations
into ECFP. 19 Sirius possesses the shortest emission wavelength among fluo-
rescent proteins reported to date and is relatively bright and pH insensitive.

2.2. Red chromophore synthesis via blue intermediates
Amino acids at position 65 affect the resulting chemical structure of the
chromophore and therefore vary widely among GFP-like proteins of vari-
ous colors. Most GFP-derived fluorescent proteins have a threonine at this
position, unlike A. victoria GFP ( Table 8.1 ), which has a serine. This substi-
tution results in proteins that have a single red-shifted excitation peak, fluo-
resce more intensely than wild type, and fold approximately four times faster
than wild-type GFP. 20 The red chromophore of the Discosoma sp . (DsRed)
fluorescent protein has an extended GFP-like core with an additional des-
aturated C N bond at the Gln65 residue (position 66 in DsRed2)

( Table 8.1 ). The red-shifted absorbance and emission are therefore provided
by an extended

a

-electron conjugation. 4 An early hypothesis in which the
maturation of the DsRed-like red chromophore was thought to proceed via

p