PDBsum entry 2a54

Luminescent protein

PDB id: 2a54

Contents

Protein chains

64 a.a. *

168 a.a. *

59 a.a. *

Metals

_CL

Waters ×574

* Residue conservation analysis

PDB id:

2a54

Name:

Luminescent protein

Title:

Fluorescent protein asfp595, a143s, on-state, 1min irradiation

Structure:

Gfp-like non-fluorescent chromoprotein fp595 chain 1. Chain: a, c. Synonym: asfp595. Engineered: yes. Gfp-like non-fluorescent chromoprotein fp595 chain 2. Chain: b, d. Synonym: asfp595. Engineered: yes. Mutation: yes

Source:

Anemonia sulcata. Snake-locks sea anemone. Organism_taxid: 6108. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Octamer (from PDB file)

Resolution:

1.45Å R-factor: 0.177 R-free: 0.200

Authors:

M.Andresen,M.C.Wahl,A.C.Stiel,F.Graeter,L.Schaefer,S.Trowitzsch, G.Weber,C.Eggeling,H.Grubmueller,S.W.Hell,S.Jakobs

Key ref:

M.Andresen et al. (2005). Structure and mechanism of the reversible photoswitch of a fluorescent protein. Proc Natl Acad Sci U S A, 102, 13070-13074. PubMed id: 16135569 DOI: 10.1073/pnas.0502772102

Date:

30-Jun-05 Release date: 16-Aug-05

Protein chain

Q9GZ28  (NFCP_ANESU) -  GFP-like non-fluorescent chromoprotein FP595 from Anemonia sulcata

Seq: 232 a.a.

Struc: 64 a.a.*

Protein chains

Q9GZ28  (NFCP_ANESU) -  GFP-like non-fluorescent chromoprotein FP595 from Anemonia sulcata

Seq: 232 a.a.

Struc: 168 a.a.*

Protein chain

Q9GZ28  (NFCP_ANESU) -  GFP-like non-fluorescent chromoprotein FP595 from Anemonia sulcata

Seq: 232 a.a.

Struc: 59 a.a.

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

DOI no: 10.1073/pnas.0502772102

Proc Natl Acad Sci U S A 102:13070-13074 (2005)

PubMed id: 16135569

Structure and mechanism of the reversible photoswitch of a fluorescent protein.

M.Andresen, M.C.Wahl, A.C.Stiel, F.Gräter, L.V.Schäfer, S.Trowitzsch, G.Weber, C.Eggeling, H.Grubmüller, S.W.Hell, S.Jakobs.

ABSTRACT

Proteins that can be reversibly photoswitched between a fluorescent and a nonfluorescent state bear enormous potential in diverse fields, such as data storage, in vivo protein tracking, and subdiffraction resolution light microscopy. However, these proteins could hitherto not live up to their full potential because the molecular switching mechanism is not resolved. Here, we clarify the molecular photoswitching mechanism of asFP595, a green fluorescent protein (GFP)-like protein that can be transferred from a nonfluorescent "off" to a fluorescent "on" state and back again, by green and blue light, respectively. To this end, we establish reversible photoswitching of fluorescence in whole protein crystals and show that the switching kinetics in the crystal is identical with that in solution. Subsequent x-ray analysis demonstrated that upon the absorption of a green photon, the chromophore isomerizes from a trans (off) to a cis (on) state. Molecular dynamics calculations suggest that isomerization occurs through a bottom hula twist mechanism with concomitant rotation of both bonds of the chromophoric methine ring bridge. This insight into the switching mechanism should facilitate the targeted design of photoswitchable proteins. Reversible photoswitching of the protein chromophore system within intact crystals also constitutes a step toward the use of fluorescent proteins in three-dimensional data recording.

Selected figure(s)

Figure 1.
Fig. 1. Overall structure of asFP595. A schematic ribbon representation of the quaternary tetrameric structure of asFP595 shows the four molecules in different colors and the chromophores highlighted in red.

Figure 5.
Fig. 5. MD simulations of the switching mechanism in asFP595. (a) The two possible isomerization mechanisms. (b) Forces due to the protein matrix opposing chromophore isomerization were calculated by nonequilibrium force-probe MD simulations; 10 trajectories were averaged for the four possible pathways (solid lines). For the rotate mechanism, the MYG p-hydroxyphenyl ring can either rotate toward the initially coplanar H197 (Rtop) or toward the other side (Rbot). Likewise, during HT isomerization, the bridging methine group can move along a top (HTtop) or bottom (HTbot) pathway. Control simulations of the chromophore in water show similar forces for the R and HT mechanisms (dashed curves). (Inset) MYG chromophore and relevant torsion angles. (c) Spontaneous trans-cis isomerization during free excited state MD simulations for the chromophore within the protein matrix (Upper) and in water (Lower), monitored through the dihedral angles (dashed curves) and (solid curves). The protein favors the HTbot mechanism, with both dihedral angles rotating simultaneously within a narrow time frame. Simulation of protein-free isomerization of the chromophore in water also follows HT; both directions (HTtop and HTbot) are observed in this case.

Figures were selected by an automated process.