example, replacement of Tyr-66 with Leu results in a colorless variant
that represents a trapped intermediate of the pathway. The necessity of
protein folding producing a tight conformation of Ser-65 and Gly-67 for
the cyclization step was demonstrated by the properties of another color-
less variant carrying the Ser-65-to-Gly and Tyr-66-to-Gly substitutions
under anaerobic conditions.
Formation of the chromophore is a complex mechanism involving steps
such as pre-organization of the protein, electrophilic and base catalysis, and
the subsequent slow proton abstraction. The autocatalytic modifications
that produce the cofactor in GFP are intrinsic properties as the formation
requires only a single gene in contrast to the biosynthetic pathways required
for assembly of cofactors in other proteins. Understanding the role of each
amino acid residue may have a broad relevance for other proteins with
similar chromophore-formation mechanisms that are identified not only
through biochemical analysis but also by sequence-specific searches of
the genomes of different organisms. The mechanism that produces the
chromophore in GFP is not unique to this protein as the identification of
proteins with prosthetic groups generated by post-translational modifica-
tions has expanded tremendously in recent years. The post-translational
oxidation of tyrosine residues often plays an essential role, with examples
being the formation of topaquinone in copper amine oxidases and the
cross-linking of tyrosine to cysteine in galactose oxidase. In contrast to
GFP, the spontaneous formation of such cofactors is usually facilitated by
the presence of a metal cofactor. Whereas the general scheme for cofactor
formation is known for GFP and these other proteins, the detailed mechan-
ism is still under active investigation in a number of laboratories.
Fluorescence resonance energy transfer
Fluorescence resonance energy transfer, or FRET, is a non-radiative pro-
cess by which energy from an excited energy donor, D*, is transferred to an
acceptor fluorphore, A, resulting in anexcited acceptor, A*, and a ground-state
donor, D:
D +A ↔D +A (19.1)
The rate of the excitation transfer can be written in terms of a transition
involving the wa 9 efunction of the acceptor ψA and excited donor,ψD*. The
operator for the transition is given by the product of the transition dipole
moments of the donor and acceptor divided by the separation between the
donor and acceptor,rDA, cubed:
(19.2)
k
r
∝ *
⎛
⎝
⎜⎜
⎞
⎠
⎟⎟
⎡
⎣
⎢
⎢
⎤
⎦
⎥
∫ ⎥
ψ
μμ
D DAψτ
DA
3 Ad
2
410 PART 3 UNDERSTANDING BIOLOGICAL SYSTEMS USING PHYSICAL CHEMISTRY
