PDBsum entry 3kcs

Fluorescent protein

PDB id: 3kcs

Contents

Protein chain

215 a.a. *

Waters ×303

* Residue conservation analysis

PDB id:

3kcs

Name:

Fluorescent protein

Title:

Crystal structure of pamcherry1 in the dark state

Structure:

Pamcherry1 protein. Chain: a. Engineered: yes

Source:

Discosoma sp.. Organism_taxid: 86600. Gene: pamcherry1. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.50Å R-factor: 0.174 R-free: 0.206

Authors:

V.N.Malashkevich,F.V.Subach,W.D.Zencheck,H.Xiao,G.S.Filonov,S.C.Almo, V.V.Verkhusha

Key ref:

F.V.Subach et al. (2009). Photoactivation mechanism of PAmCherry based on crystal structures of the protein in the dark and fluorescent states. Proc Natl Acad Sci U S A, 106, 21097-21102. PubMed id: 19934036 DOI: 10.1073/pnas.0909204106

Date:

21-Oct-09 Release date: 17-Nov-09

Protein chain

D1MPT3  (D1MPT3_DISSP) -  PAmCherry1 protein from Discosoma sp.

Seq: 236 a.a.

Struc: 215 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DOI no: 10.1073/pnas.0909204106

Proc Natl Acad Sci U S A 106:21097-21102 (2009)

PubMed id: 19934036

Photoactivation mechanism of PAmCherry based on crystal structures of the protein in the dark and fluorescent states.

F.V.Subach, V.N.Malashkevich, W.D.Zencheck, H.Xiao, G.S.Filonov, S.C.Almo, V.V.Verkhusha.

ABSTRACT

Photoactivatable fluorescent proteins (PAFPs) are required for super-resolution imaging of live cells. Recently, the first red PAFP, PAmCherry1, was reported, which complements the photo-activatable GFP by providing a red super-resolution color. PAmCherry1 is originally "dark" but exhibits red fluorescence after UV-violet light irradiation. To define the structural basis of PAmCherry1 photoactivation, we determined its crystal structure in the dark and red fluorescent states at 1.50 A and 1.65 A, respectively. The non-coplanar structure of the chromophore in the dark PAmChery1 suggests the presence of an N-acylimine functionality and a single non-oxidized C(alpha)-C(beta) bond in the Tyr-67 side chain in the cyclized Met-66-Tyr-67-Gly-68 tripeptide. MS data of the chromophore-bearing peptide indicates the loss of 20 Da upon maturation, whereas tandem MS reveals the C(alpha)-N bond in Met-66 is oxidized. These data indicate that PAmCherry1 in the dark state possesses the chromophore N-[(E)-(5-hydroxy-1H-imidazol-2-yl)methylidene]acetamide, which, to our knowledge, has not been previously observed in PAFPs. The photoactivated PAmCherry1 exhibits a non-coplanar anionic DsRed-like chromophore but in the trans configuration. Based on the crystallographic analysis, MS data, and biochemical analysis of the PAmCherry1 mutants, we propose the detailed photoactivation mechanism. In this mechanism, the excited-state PAmCherry1 chromophore acts as the oxidant to release CO(2) molecule from Glu-215 via a Koble-like radical reaction. The Glu-215 decarboxylation directs the carbanion formation resulting in the oxidation of the Tyr-67 C(alpha)-C(beta) bond. The double bond extends the pi-conjugation between the phenolic ring of Tyr-67, the imidazolone, and the N-acylimine, resulting in the red fluorescent chromophore.

Selected figure(s)

Figure 2.
Structures of the PAmCherry1 chromophore within its environment in the OFF (A) and ON (B) states. Water molecules are shown as red spheres; hydrogen bonds are shown as green dashed lines. The mCherry chromophore and its environment (PDB ID code 2H5Q) are shown for comparison (C).

Figure 3.
Suggested mechanisms for the formation of the PAmCherry1 dark chromophore (OFF state) and its light-induced conversion into the fluorescent state (ON state) are shown. The cyclized form is the chromophore with the non-oxidized bond between p-hydroxyphenyl and imidazolone moieties and without N-acylimine. Hydrogen bonds are shown with dashed lines. Intermediate compounds are shown in parentheses. The chromophore in the excited state is denoted with asterisk. hv, indicates the illumination with violet light. Migration of the electron density is shown with curved arrows.

Figures were selected by an automated process.