BIOCHEMISTRY OF BIOLUMINESCENCE IN AEQUORIN

  • Prendergast, Franklyn G (PI)

Project: Research project

Project Details

Description

The jelllyfish Aequorea forskalea emits bright green bioluminescence when
stimulated mechanically, electrically or chemically. Two proteins are
primarily responsible for the light. One is aequorin, the actual source of
the bioluminescence (whose emission maximum is at 469 nm) and the other is
a green fluorescent protein (GFP) which accepts energy from aequorin (in
vivo) and emits green light (Lambda max 510 nm). The sole requirement for
aequorin bioluminescence is the presence of Ca++ (or a variety of other
divalent and trivalent metal ions including the lanthanides). A substrate
(luciferin) and oxygen are trapped within the protein; the binding of Ca++
to the protein inspires a rapid catalytic oxidation of the luciferin by
some form of oxygen which results in emission of light. After being thus
discharged, the protein acquires a bright blue fluorescence. Aequorin
turns over only once in response to binding Ca++. The green fluorescent
protein structurally extremely stable and the fluorophore is an intrinsic
part of the protein is backbone presumably arising by a unique
post-translational modification. For the next grant period we propose to
determine how the protein binds oxygen and to investigate the
intramolecular mechanisms of oxidation of the luciferin. We will then do a
detailed study of the photophysics of the fluorescence processes of both
aequorin and GFP and investigate the mechanisms underlying bioluminescence
energy transfer. We will use fluorescence spectroscopic methods to probe
the dipolar character of the environment of the fluorophore in both
proteins and to determine the dynamics of the fluorophores through
measurements of time resolved anisotropy in the picosecond domain by use of
multiple frequency phase fluorometry. Luminescence lifetime and circular
polarized luminescence measurements of Tb(III) and Eu(III) bound to
aequorin will be used to determine the coordination chemistry at the
lanthanide binding sites. We also propose to determine the aminoacid
sequence of GFP and to investigate the biochemical mechanisms mediating the
post-translational synthesis of the green fluorescent moiety.
StatusFinished
Effective start/end date7/1/8212/31/88

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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