| Rebekka M. Wachter Research |
Chromophore biosynthesis in fluorescent
proteins
The catalytic and structural roles of proteins in biological
organisms are tightly coupled to the primary sequence of their
constituent amino acid building blocks, which determines the
three-dimensional fold of the protein. However, the
conservative nature of the genetic code limits the available
amino acids from which proteins may be constructed to only
twenty different kinds. To expand the repertoire of accessible
biochemical properties, some proteins undergo covalent
modifications once the correct tertiary fold has been attained.
proteins
The catalytic and structural roles of proteins in biological
organisms are tightly coupled to the primary sequence of their
constituent amino acid building blocks, which determines the
three-dimensional fold of the protein. However, the
conservative nature of the genetic code limits the available
amino acids from which proteins may be constructed to only
twenty different kinds. To expand the repertoire of accessible
biochemical properties, some proteins undergo covalent
modifications once the correct tertiary fold has been attained.
Department of Chemistry and Biochemistry
Center for Bioenergy and Photosynthesis
Center for Bioenergy and Photosynthesis
Protein Maturation
Protein self-processing reactions are reactions where the
catalytic groups and substrate are part of the same protein
chain, such that both are encoded for by a single gene. An
important class of covalent alterations concerns the
autocatalytic oxidation of tyrosine residues, as in the
biogenesis of quinone-type redox cofactors. However, a
highly unusual metal-independent tyrosine oxidation process
is exemplified by a group of colored proteins whose founding
member is green fluorescent protein (GFP). A
tyrosine-bearing tripeptide located in the center of the
eleven-stranded beta barrel is spontaneously modified to
yield a chromogenic entity that is responsible for the colorful
appearance of these proteins. Thus, the brightly fluorescing
chromophores that are the trademark of GFP-like proteins
are the result of an oxidative tyrosine modification mediated
by a peptide cyclization reaction rather than metallochemistry.
catalytic groups and substrate are part of the same protein
chain, such that both are encoded for by a single gene. An
important class of covalent alterations concerns the
autocatalytic oxidation of tyrosine residues, as in the
biogenesis of quinone-type redox cofactors. However, a
highly unusual metal-independent tyrosine oxidation process
is exemplified by a group of colored proteins whose founding
member is green fluorescent protein (GFP). A
tyrosine-bearing tripeptide located in the center of the
eleven-stranded beta barrel is spontaneously modified to
yield a chromogenic entity that is responsible for the colorful
appearance of these proteins. Thus, the brightly fluorescing
chromophores that are the trademark of GFP-like proteins
are the result of an oxidative tyrosine modification mediated
by a peptide cyclization reaction rather than metallochemistry.
During the last few years, our group has made substantial progress in
shedding light on the mechanism of color acquisition in green fluorescent
protein. A combination of protein X-ray crystallographic and kinetic
experiments has lead to the development of a mechanistic model that
entails conformational pre-organization, electrophilic and base catalysis,
and the production of hydrogen peroxide upon protein oxidation. This
process is concluded by a slow proton abstraction step from a
tyrosine-derived carbon acid. The reduction of molecular oxygen to
hydrogen peroxide is the major rate-limiting step and proceeds on a
relatively slow time scale, opening the door to time-resolved structural
studies that utilize X-ray crystallography to investigate cryo-trapped
intermediates.
shedding light on the mechanism of color acquisition in green fluorescent
protein. A combination of protein X-ray crystallographic and kinetic
experiments has lead to the development of a mechanistic model that
entails conformational pre-organization, electrophilic and base catalysis,
and the production of hydrogen peroxide upon protein oxidation. This
process is concluded by a slow proton abstraction step from a
tyrosine-derived carbon acid. The reduction of molecular oxygen to
hydrogen peroxide is the major rate-limiting step and proceeds on a
relatively slow time scale, opening the door to time-resolved structural
studies that utilize X-ray crystallography to investigate cryo-trapped
intermediates.
| Chromophore intermediate state trapped by mutagenesis. |
Though GFP is the best-studied member of this group of homologous
proteins, most members are vividly fluorescent and their colors cover a
broad range of the visible spectrum (cyan, green, yellow, red,
non-fluorescent purple). As complete maturation requires only a single
gene product, the diversity of biotechnological applications of GFP-like
proteins as fluorescent markers and biosensors has exploded during the
last decade. However, our fundamental understanding of the biosynthetic
mechanisms yielding the currently known set of chemically distinct
fluorophores continues to lag behind the development of novel
technologies useful for tracking cellular events in real time.
proteins, most members are vividly fluorescent and their colors cover a
broad range of the visible spectrum (cyan, green, yellow, red,
non-fluorescent purple). As complete maturation requires only a single
gene product, the diversity of biotechnological applications of GFP-like
proteins as fluorescent markers and biosensors has exploded during the
last decade. However, our fundamental understanding of the biosynthetic
mechanisms yielding the currently known set of chemically distinct
fluorophores continues to lag behind the development of novel
technologies useful for tracking cellular events in real time.
| Progress curves for intermediates and products generated during chromophore maturation. |
In addition, we have recently expanded the scope of this project to include directed evolution of
fluorescent proteins (in collaboration with Dr. Neal Woodbury, ASU Chemistry and the Biodesign
Institute), and the investigation of photochemical processes by ultra-fast spectroscopy (in
collaboration with Dr. Su Lin at ASU and Dr. Peter J. Tonge at SUNY Stony Brook).
fluorescent proteins (in collaboration with Dr. Neal Woodbury, ASU Chemistry and the Biodesign
Institute), and the investigation of photochemical processes by ultra-fast spectroscopy (in
collaboration with Dr. Su Lin at ASU and Dr. Peter J. Tonge at SUNY Stony Brook).
| GFP-like fluorescent proteins bear similar, yet chemically distinct chromophores that are synthesized from three internal residues. |