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Crystallographic structures of Discosoma red fluorescent protein with immature and mature chromophores: linking peptide bond trans-cis isomerization and acylimine formation in chromophore maturation.
Biochemistry. 2005 Jul 26; 44(29):9833-40.B

Abstract

The mature self-synthesizing p-hydroxybenzylideneimidazolinone-like fluorophores of Discosoma red fluorescent protein (DsRed) and Aequorea victoria green fluorescent protein (GFP) are extensively studied as powerful biological markers. Yet, the spontaneous formation of these fluorophores by cyclization, oxidation, and dehydration reactions of tripeptides within their protein environment remains incompletely understood. The mature DsRed fluorophore (Gln 66, Tyr 67, and Gly 68) differs from the GFP fluorophore by an acylimine that results in Gln 66 Calpha planar geometry and by a Phe 65-Gln 66 cis peptide bond. DsRed green-to-red maturation includes a green-fluorescing immature chromophore and requires a chromophore peptide bond trans-cis isomerization that is slow and incomplete. To clarify the unique structural chemistry for the individual immature "green" and mature "red" chromophores of DsRed, we report here the determination and analysis of crystal structures for the wild-type protein (1.4 A resolution), the entirely green DsRed K70M mutant protein (1.9 A resolution), and the DsRed designed mutant Q66M (1.9 A resolution), which shows increased red chromophore relative to the wild-type DsRed. Whereas the mature, red-fluorescing chromophore has the expected cis peptide bond and a sp(2)-hybridized Gln 66 Calpha with planar geometry, the crystal structure of the immature green-fluorescing chromophore of DsRed, presented here for the first time, reveals a trans peptide bond and a sp(3)-hybridized Gln 66 Calpha with tetrahedral geometry. These results characterize a GFP-like immature green DsRed chromophore structure, reveal distinct mature and immature chromophore environments, and furthermore provide evidence for the coupling of acylimine formation with trans-cis isomerization.

Authors+Show Affiliations

Department of Molecular Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

16026155

Citation

Tubbs, Julie L., et al. "Crystallographic Structures of Discosoma Red Fluorescent Protein With Immature and Mature Chromophores: Linking Peptide Bond Trans-cis Isomerization and Acylimine Formation in Chromophore Maturation." Biochemistry, vol. 44, no. 29, 2005, pp. 9833-40.
Tubbs JL, Tainer JA, Getzoff ED. Crystallographic structures of Discosoma red fluorescent protein with immature and mature chromophores: linking peptide bond trans-cis isomerization and acylimine formation in chromophore maturation. Biochemistry. 2005;44(29):9833-40.
Tubbs, J. L., Tainer, J. A., & Getzoff, E. D. (2005). Crystallographic structures of Discosoma red fluorescent protein with immature and mature chromophores: linking peptide bond trans-cis isomerization and acylimine formation in chromophore maturation. Biochemistry, 44(29), 9833-40.
Tubbs JL, Tainer JA, Getzoff ED. Crystallographic Structures of Discosoma Red Fluorescent Protein With Immature and Mature Chromophores: Linking Peptide Bond Trans-cis Isomerization and Acylimine Formation in Chromophore Maturation. Biochemistry. 2005 Jul 26;44(29):9833-40. PubMed PMID: 16026155.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Crystallographic structures of Discosoma red fluorescent protein with immature and mature chromophores: linking peptide bond trans-cis isomerization and acylimine formation in chromophore maturation. AU - Tubbs,Julie L, AU - Tainer,John A, AU - Getzoff,Elizabeth D, PY - 2005/7/20/pubmed PY - 2005/10/19/medline PY - 2005/7/20/entrez SP - 9833 EP - 40 JF - Biochemistry JO - Biochemistry VL - 44 IS - 29 N2 - The mature self-synthesizing p-hydroxybenzylideneimidazolinone-like fluorophores of Discosoma red fluorescent protein (DsRed) and Aequorea victoria green fluorescent protein (GFP) are extensively studied as powerful biological markers. Yet, the spontaneous formation of these fluorophores by cyclization, oxidation, and dehydration reactions of tripeptides within their protein environment remains incompletely understood. The mature DsRed fluorophore (Gln 66, Tyr 67, and Gly 68) differs from the GFP fluorophore by an acylimine that results in Gln 66 Calpha planar geometry and by a Phe 65-Gln 66 cis peptide bond. DsRed green-to-red maturation includes a green-fluorescing immature chromophore and requires a chromophore peptide bond trans-cis isomerization that is slow and incomplete. To clarify the unique structural chemistry for the individual immature "green" and mature "red" chromophores of DsRed, we report here the determination and analysis of crystal structures for the wild-type protein (1.4 A resolution), the entirely green DsRed K70M mutant protein (1.9 A resolution), and the DsRed designed mutant Q66M (1.9 A resolution), which shows increased red chromophore relative to the wild-type DsRed. Whereas the mature, red-fluorescing chromophore has the expected cis peptide bond and a sp(2)-hybridized Gln 66 Calpha with planar geometry, the crystal structure of the immature green-fluorescing chromophore of DsRed, presented here for the first time, reveals a trans peptide bond and a sp(3)-hybridized Gln 66 Calpha with tetrahedral geometry. These results characterize a GFP-like immature green DsRed chromophore structure, reveal distinct mature and immature chromophore environments, and furthermore provide evidence for the coupling of acylimine formation with trans-cis isomerization. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/16026155/Crystallographic_structures_of_Discosoma_red_fluorescent_protein_with_immature_and_mature_chromophores:_linking_peptide_bond_trans_cis_isomerization_and_acylimine_formation_in_chromophore_maturation_ L2 - https://dx.doi.org/10.1021/bi0472907 DB - PRIME DP - Unbound Medicine ER -