RENILLA
GFP: DISCOVERED AND IDENTIFIED BY
FUNCTION.
John E. Wampler, Dept. of Biochemistry
and Molecular Biology, University of Georgia,
Life Sciences Bldg., Athens, GA 30602-7229.
Prior to the 70's, spectral data and
observations of living animals by Cormier,
Hastings, Johnson, Morin and their colleagues
revealed intriguing spectral differences in many
cases between in vitro bioluminescence reactions
of coelenterate systems and the in vivo bio-
luminescence. In '67 Johnson proposed that a
green fluorescent protein (GFP)isolated from
Aequorea might be responsible for this
difference by energy transfer from the
blue-light emitting photoprotein. During the
early 70's these observations prompted several
projects in the various laboratories to identify
the emitters involved. The first step, spectral
identity, required a number of measurement
improvements pioneered by earlier spectral
studies by McElroy, Reynolds and Seliger.
Before 1971, we were slow to recognize and
define the Renilla green fluorescent emitter as
a separate protein. Indeed, its spectroscopy was
much better defined at that time than was the
protein itself. The key to recognizing it as a
separate protein lay in the discovery of
increases in quantum yield along with changes
in the spectrum of in vitro reactions at high
protein concentration. In late 1971, we were
finally able to separate, purify and
characterize Renilla GFP (later fully
characterized by Ward and Cormier) and
reconstruct a homogeneous in vitro system with
emission matching that of the living organism.
The non-radiative energy transfer mechanism was
unambiguously demonstrated by these latter
studies showing its dependence on luciferase-GFP
interactions and by studies of marked quantum
yield increases when "dark" luciferin analogs
synthesized by Russ Hart in 1978 were used.
The properties of Renilla GFP, which
distinguished it from Aequorea GFP in those
early studies and which continue to suggest
important differences in both structure and
function, present a challenge to modern
structural studies to explain their basis.