BioPHYSICAL chemistry

most commonly used GFPs is enhanced

GFP, which produces a larger amount of

fluorescence due to an improvement in its

quantum efficiency, resulting from changes

in the protein surrounding the pigment.

Mechanism of chromophore formation

The spectrum of the wild-type Aequoreapro-

tein shows the presence of two bands, with

a major peak at 395 nm and a minor peak

at 475 nm, suggesting the presence of more

than one optical species. The optical spectrum

is more complex at low temperature, with

the absorption bands showing significantly

more structure, indicating the presence

of several trapped states that are close in

energy. Using transient spectroscopy, the

kinetics of the optical spectrum after excita-

tion were shown to have a complex behavior. To investigate the reason

for the two peaks, the optical properties of the chromophore have been

probed. For example, significant spectral shifts are evident for a variant

that is identified as YFP, or yellow fluorescent protein, which has one of

the longest-wavelength emissions of all engineered variants. A pH titra-

tion of this protein shows a systematic behavior which can be modeled

as arising from a pKAassociated with the chromophore (Figure 19.5).

The simplest interpretation is that the absorbance at 475 nm arises from

GFPs that contain a deprotonated chromophore and that the 395-nm peak

arises from those GFPs containing a protonated chromophore. These and

other observations lead to a model that the chromophore of GFP has two

states formed during the cofactor formation. The requirement of oxygen

for fluorophore formation suggested that the involvement of oxygen

represented the rate-limiting step. The folding of the peptide chain into the

barrel establishes the protein environment surrounding residues Ser-65–

Tyr-66 – Gly-67, which initiates formation (Figure 19.6). Then cyclization

occurs due to nucleophilic attack of the amide of Gly-67 on the carbonyl

of Ser-65 followed by dehydration. The resulting conjugated molecule gives

GFP its color and fluorescence for the first time. For DsRed, there is an

additional oxidation step that leads to the extension of the conjugation.

The resulting chromophore has an absorbance at a longer wavelength, as

would be predicted using a particle-in-a-box model (Chapter 10).

The delineation of the cofactor-formation mechanism including the

identification of the intermediates is being performed through a com-

bination of functional and structural studies of mutants. Mutants that

are defective because of changes that stop the assembly at intermediate

points provide the opportunity to investigate intermediate states. For

408 PART 3 UNDERSTANDING BIOLOGICAL SYSTEMS USING PHYSICAL CHEMISTRY

O

O

R

R

O

O RO

O

O

OO

R

RR

SOL

N

SOL

HO SOL

SOL

O

O

N

N

RR

R

R

N

N

N

N

SOL N

N

N

N

V 68

2.9

2.7

3.0 2.6

3.0

3.1

3.02.6

2.6

2.9

2.6

3.0

2.8

2.9

3.0

2.6 2.6

2.5

3.2

3.0

S 205 E 222

N 146

H 148

T 203

T 62

R 96

Q 183

Q 94

Q 69



Figure 19.4A schematic diagram showing the
protein interactions between the chromophore and
the protein environment for wild-type GFP. Modified
from Wachter et al. (1997).