Reaction progress of chromophore biogenesis in green fluorescent protein

Liping Zhang, Hetal N. Patel, Jason W. Lappe, Rebekka Wachter

Research output: Contribution to journalArticle

76 Citations (Scopus)

Abstract

The mature form of green fluorescent protein (GFP) is generated by a spontaneous self-modification process that is essentially irreversible. A key step in chromophore biosynthesis involves slow air oxidation of an intermediate species, in which the backbone atoms of residues 65-67 have condensed to form a five-membered heterocycle. We have investigated the kinetics of hydrogen peroxide evolution during in vitro GFP maturation and found that the H 2O2 coproduct is generated prior to the acquisition of green fluorescence at a stoichiometry of 1:1 (peroxide/chromophore). The experimental progress curves were computer-fitted to a three-step mechanism, in which the first step proceeds with a time constant of 1.5 (±1.1) min and includes protein folding and peptide cyclization. Kinetic data obtained by HPLC analysis support a rapid cyclization reaction that can be reversed upon acid denaturation. The second step proceeds with a time constant of 34.0 (±1.5) min and entails rate-limiting protein oxidation, as supported by a mass loss of 2 Da observed for tryptic peptides derived from species that accumulate during the reaction. The final step in GFP maturation proceeds with a time constant of 10.6 (±1.2) min, suggesting that this step may contribute to overall rate retardation. We propose that under highly aerobic conditions, the dominant reaction path follows a cyclization-oxidation- dehydration mechanism, in which dehydration of the heterocycle is facilitated by slow proton abstraction from the Tyr66 β-carbon. In combination, the results presented here suggest a role for molecular oxygen in trapping the cyclized form of GFP.

Original languageEnglish (US)
Pages (from-to)4766-4772
Number of pages7
JournalJournal of the American Chemical Society
Volume128
Issue number14
DOIs
StatePublished - Apr 12 2006

Fingerprint

Chromophores
Green Fluorescent Proteins
Cyclization
Proteins
Dehydration
Oxidation
Peptides
Protein folding
Denaturation
Kinetics
Molecular oxygen
Biosynthesis
Protein Folding
Peroxides
Stoichiometry
Hydrogen Peroxide
Protons
Carbon
Hydrogen peroxide
Fluorescence

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Reaction progress of chromophore biogenesis in green fluorescent protein. / Zhang, Liping; Patel, Hetal N.; Lappe, Jason W.; Wachter, Rebekka.

In: Journal of the American Chemical Society, Vol. 128, No. 14, 12.04.2006, p. 4766-4772.

Research output: Contribution to journalArticle

Zhang, Liping ; Patel, Hetal N. ; Lappe, Jason W. ; Wachter, Rebekka. / Reaction progress of chromophore biogenesis in green fluorescent protein. In: Journal of the American Chemical Society. 2006 ; Vol. 128, No. 14. pp. 4766-4772.
@article{ae118b8fec66417489a3efbff90fb88b,
title = "Reaction progress of chromophore biogenesis in green fluorescent protein",
abstract = "The mature form of green fluorescent protein (GFP) is generated by a spontaneous self-modification process that is essentially irreversible. A key step in chromophore biosynthesis involves slow air oxidation of an intermediate species, in which the backbone atoms of residues 65-67 have condensed to form a five-membered heterocycle. We have investigated the kinetics of hydrogen peroxide evolution during in vitro GFP maturation and found that the H 2O2 coproduct is generated prior to the acquisition of green fluorescence at a stoichiometry of 1:1 (peroxide/chromophore). The experimental progress curves were computer-fitted to a three-step mechanism, in which the first step proceeds with a time constant of 1.5 (±1.1) min and includes protein folding and peptide cyclization. Kinetic data obtained by HPLC analysis support a rapid cyclization reaction that can be reversed upon acid denaturation. The second step proceeds with a time constant of 34.0 (±1.5) min and entails rate-limiting protein oxidation, as supported by a mass loss of 2 Da observed for tryptic peptides derived from species that accumulate during the reaction. The final step in GFP maturation proceeds with a time constant of 10.6 (±1.2) min, suggesting that this step may contribute to overall rate retardation. We propose that under highly aerobic conditions, the dominant reaction path follows a cyclization-oxidation- dehydration mechanism, in which dehydration of the heterocycle is facilitated by slow proton abstraction from the Tyr66 β-carbon. In combination, the results presented here suggest a role for molecular oxygen in trapping the cyclized form of GFP.",
author = "Liping Zhang and Patel, {Hetal N.} and Lappe, {Jason W.} and Rebekka Wachter",
year = "2006",
month = "4",
day = "12",
doi = "10.1021/ja0580439",
language = "English (US)",
volume = "128",
pages = "4766--4772",
journal = "Journal of the American Chemical Society",
issn = "0002-7863",
publisher = "American Chemical Society",
number = "14",

}

TY - JOUR

T1 - Reaction progress of chromophore biogenesis in green fluorescent protein

AU - Zhang, Liping

AU - Patel, Hetal N.

AU - Lappe, Jason W.

AU - Wachter, Rebekka

PY - 2006/4/12

Y1 - 2006/4/12

N2 - The mature form of green fluorescent protein (GFP) is generated by a spontaneous self-modification process that is essentially irreversible. A key step in chromophore biosynthesis involves slow air oxidation of an intermediate species, in which the backbone atoms of residues 65-67 have condensed to form a five-membered heterocycle. We have investigated the kinetics of hydrogen peroxide evolution during in vitro GFP maturation and found that the H 2O2 coproduct is generated prior to the acquisition of green fluorescence at a stoichiometry of 1:1 (peroxide/chromophore). The experimental progress curves were computer-fitted to a three-step mechanism, in which the first step proceeds with a time constant of 1.5 (±1.1) min and includes protein folding and peptide cyclization. Kinetic data obtained by HPLC analysis support a rapid cyclization reaction that can be reversed upon acid denaturation. The second step proceeds with a time constant of 34.0 (±1.5) min and entails rate-limiting protein oxidation, as supported by a mass loss of 2 Da observed for tryptic peptides derived from species that accumulate during the reaction. The final step in GFP maturation proceeds with a time constant of 10.6 (±1.2) min, suggesting that this step may contribute to overall rate retardation. We propose that under highly aerobic conditions, the dominant reaction path follows a cyclization-oxidation- dehydration mechanism, in which dehydration of the heterocycle is facilitated by slow proton abstraction from the Tyr66 β-carbon. In combination, the results presented here suggest a role for molecular oxygen in trapping the cyclized form of GFP.

AB - The mature form of green fluorescent protein (GFP) is generated by a spontaneous self-modification process that is essentially irreversible. A key step in chromophore biosynthesis involves slow air oxidation of an intermediate species, in which the backbone atoms of residues 65-67 have condensed to form a five-membered heterocycle. We have investigated the kinetics of hydrogen peroxide evolution during in vitro GFP maturation and found that the H 2O2 coproduct is generated prior to the acquisition of green fluorescence at a stoichiometry of 1:1 (peroxide/chromophore). The experimental progress curves were computer-fitted to a three-step mechanism, in which the first step proceeds with a time constant of 1.5 (±1.1) min and includes protein folding and peptide cyclization. Kinetic data obtained by HPLC analysis support a rapid cyclization reaction that can be reversed upon acid denaturation. The second step proceeds with a time constant of 34.0 (±1.5) min and entails rate-limiting protein oxidation, as supported by a mass loss of 2 Da observed for tryptic peptides derived from species that accumulate during the reaction. The final step in GFP maturation proceeds with a time constant of 10.6 (±1.2) min, suggesting that this step may contribute to overall rate retardation. We propose that under highly aerobic conditions, the dominant reaction path follows a cyclization-oxidation- dehydration mechanism, in which dehydration of the heterocycle is facilitated by slow proton abstraction from the Tyr66 β-carbon. In combination, the results presented here suggest a role for molecular oxygen in trapping the cyclized form of GFP.

UR - http://www.scopus.com/inward/record.url?scp=33646026448&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33646026448&partnerID=8YFLogxK

U2 - 10.1021/ja0580439

DO - 10.1021/ja0580439

M3 - Article

C2 - 16594713

AN - SCOPUS:33646026448

VL - 128

SP - 4766

EP - 4772

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

SN - 0002-7863

IS - 14

ER -