Scripps VIVO scripps research logo

  • Index
  • Log in
  • Home
  • People
  • Organizations
  • Research
  • Events
Search form

Structural evidence for an enolate intermediate in gfp fluorophore biosynthesis

Academic Article
uri icon
  • Overview
  • Identity
  • Additional Document Info
  • View All
scroll to property group menus

Overview

authors

  • Barondeau, D. P.
  • Tainer, John
  • Getzoff, Elizabeth

publication date

  • March 2006

journal

  • Journal of the American Chemical Society  Journal

abstract

  • The Aequorea victoria green fluorescent protein (GFP) creates a fluorophore from its component amino acids Ser65, Tyr66, and Gly67 through a remarkable post-translational modification, involving spontaneous peptide backbone cyclization, dehydration, and oxidation reactions. Here we test and extend the understanding of fluorophore biosynthesis by coupling chemical reduction and anaerobic methodologies with kinetic analyses and protein structure determination. Two high-resolution structures of dithionite-treated GFP variants reveal a previously uncharacterized enolate intermediate form of the chromophore that is viable in generating a fluorophore (t1/2 = 39 min-1) upon exposure to air. Isolation of this enolate intermediate will now allow specific probing of the rate-limiting oxidation step for fluorophore biosynthesis in GFP and its red fluorescent protein homologues. Such targeted characterizations may lead to the design of faster maturing proteins with enhanced applications in biotechnology and cell biology. Moreover, our results reveal how the GFP protein environment mimics enzyme systems, by stabilizing an otherwise high energy enolate intermediate to achieve its post-translational modification.

subject areas

  • Crystallography, X-Ray
  • Glycine
  • Green Fluorescent Proteins
  • Kinetics
  • Models, Molecular
  • Protein Conformation
  • Protein Processing, Post-Translational
  • Protein Structure, Secondary
  • Serine
  • Tyrosine
scroll to property group menus

Identity

International Standard Serial Number (ISSN)

  • 0002-7863

Digital Object Identifier (DOI)

  • 10.1021/ja0552693

PubMed ID

  • 16522096
scroll to property group menus

Additional Document Info

start page

  • 3166

end page

  • 3168

volume

  • 128

issue

  • 10

©2021 The Scripps Research Institute | Terms of Use | Powered by VIVO

  • About
  • Contact Us
  • Support