BioPHYSICAL chemistry

synthesis of the pigment from the polypeptide chain. Digestion

of GFP showed that the cofactor is formed by cyclization of

residues Ser-65, Tyr-66, and Gly-67 (Figure 19.1), consistent

with the observation of changes in the fluorescence when

those amino acid residues were changed by mutagenesis. After

formation the pigment is attached to the protein and, as a result,

expression of the gene coding for GFP leads directly to the

appearance of the color and fluorescence.

In most cases, the gene for GFP can be attached to a gene of

interest and the resulting protein will be fluorescent, allowing

tracking of genes using molecular biology. Such an approach

was a significant improvement over the more traditional approach of label-

ing proteins with fluorescent compounds to determine their cellular

localization and possibly conformational changes. Traditionally, such

work was performed by first purifying the protein and then chemically

modifying it to attach a reactive organic fluorophore. Such labeling

studies required chemical attachment of the dye followed by reintroduc-

tion of the labeled protein into the cell. The use of GFP alleviates the

necessity of purifying and manipulating the protein, offering a signific-

ant improvement for molecular imaging in cells at both a laboratory and

industrial scale. For example, GFP has been used to optimize the expres-

sion of proteins by real-time monitoring of GFP fluorescence. GFP has

also been used to study microbial growth and dispersion of organisms in

natural environments.

The spectral range of fluorescent proteins expanded when new sources

where considered. A number of mutants of GFP were identified in muta-

genesis screens as having a color shift. The effect of specific amino acid

residues was further probed using mutagenesis. Discovery of GFP-like

proteins from the coral Anthozoasignificantly expanded the wavelength

region covered by these proteins as the new protein, DsRed, has a spec-

trum whose main absorption peak is shifted to the red compared to GFP.

These discoveries produced a set of fluorescent proteins whose spectra

ranged from green to red with emission maxima from 475 to 600 nm

(Figure 19.2).

The structures of GFP and various mutants have been solved and

shown to be highly homologous. GFP is an 11-stranded βbarrel with

an α helix that runs up the axis of the cylinder (Figure 19.3). The

cylinder has a diameter of approximately 30 Å and a length of approx-

imately 40 Å. The chromophore is attached to the αhelix and buried

in the center of the barrel. Folding of GFP into the barrel is crucial to

the formation of the chromophore and its fluorescent properties. By

enclosing the chromophore inside of the barrel, it is protected from the

aqueous environment.

In wild-type GFP, the chromophore forms a well-defined structure that

is stabilized by a hydrogen-bonding network involving the surrounding

406 PART 3 UNDERSTANDING BIOLOGICAL SYSTEMS USING PHYSICAL CHEMISTRY

φ τ

Ox

Oy

Gly67

Ser65

N

Figure 19.1Structure of the
chromophore of GFP formed
after cyclization of the amino
acid residues Ser-65, Tyr-66,
and Gly-67.