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Fig. 3 Quaternary structure of tetrameric EosFP (cartoon representation). (a) Tetramer. (b) A/B
interface. (c) A/C interface. The chromophore is highlighted in green
hydrophilic contacts (Tyr39, Glu142, Asn144, Ser147, Asn149, Tyr151, Arg168,
Asn170, Glu172, Tyr200, Ser202, Gln204, Ser208).
Natural anthozoan FPs are frequently found to form tightly binding tetramers
that can only be broken up by harsh procedures, causing irreversible denaturation of
the polypeptide chains [ 33 - 35 ]. The four protomers A-D are arranged as dimers of
dimers [ 36 ], so that two different interfaces between dimers can be distinguished
(Fig. 3a ). The A/C (B/D) interface (Fig. 3c ) is more extended than the A/B (C/D)
interface (Fig. 3b ). It is hydrophilic and stabilized by salt bridges and hydrogen
bonds between polar residues and structural waters [ 37 , 38 ]. It is reinforced by the
C-terminal end of one chain intertwined with the other. The A/B interface features a
cluster of hydrophobic amino acids encircled by polar amino acids.
3 Protein Engineering
3.1 Maturation and Thermotolerance
Wild-type GFP folds fairly efficiently when expressed at or below room tempera-
ture, but its folding efficiency declines steeply at higher temperatures. However,
once it has matured, i.e., turned into its functional form at low temperature, GFP is
thermodynamically stable and fluoresces at temperatures up to at least 65 C. For
applications that involve mammalian cell cultures, it is necessary that the FP
expresses and functions well at 37 C. Codon optimization of GFP has led to an
increase in the levels of protein expression by fourfold at 37 C[ 22 , 39 ]. By
extensive mutational studies, a number of mostly bulky amino acids have been
identified in GFP that apparently contribute to the lack of expression at higher
temperature [ 5 ]. Some of these residues are located close to the chromophore
(Phe64Leu and Ser72Ala), whereas others are more remote, either buried inside
the protein (Val163Ala) or in contact with the solvent (Met153Thr, Ser175Gly,
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