NMR derived topology of a GFP-photoprotein energy transfer complex.
Related Articles NMR derived topology of a GFP-photoprotein energy transfer complex.
J Biol Chem. 2010 Oct 6;
Authors: Titushin MS, Feng Y, Stepanyuk GA, Li Y, Markova SV, Golz S, Wang BC, Lee J, Wang J, Vysotski ES, Liu ZJ
Forster resonance energy transfer within a protein-protein complex has previously been invoked to explain emission spectral modulation observed in several bioluminescence systems. Here we present a spatial structure of a complex of the Ca2+-regulated photoprotein ?lytin with its Green-fluorescent Protein (cgGFP) from the jellyfish Clytia gregaria, and show that it accounts for the bioluminescence properties of this system in vitro. We adopted an indirect approach of combining X-ray crystallography determined structures of the separate proteins, NMR spectroscopy, computational docking and mutagenesis. Heteronuclear NMR spectroscopy using variously 15N,13C,2H-enriched proteins enabled assignment of backbone resonances of more than 94% of the residues of both proteins. In a mixture of the two proteins at millimolar concentrations, complexation was inferred from perturbations of certain 1H-15N HSQC-resonances, which could be mapped to those residues involved at the interaction site. A docking computation using HADDOCK was employed constrained by the sites of interaction, to deduce an overall spatial structure of the complex. Contacts within the clytin-cgGFP complex and electrostatic complementarity of interaction surfaces argued for a weak protein-protein complex. A weak affinity was also observed by isothermal titration calorimetry (KD = 0.9 mM). Mutation of clytin residues located at the interaction site, reduced the degree of protein-protein association concomitant with a loss of effectiveness of cgGFP in color-shifting the bioluminescence. It is suggested that this clytin-cgGFP structure corresponds to the transient complex previously postulated to account for the energy transfer effect of GFP in the bioluminescence of aequorin or Renilla luciferase.
PMID: 20926380 [PubMed - as supplied by publisher]
Source:
PubMed