Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-angstrom resolution

The crystal structure of DsRed, a red fluorescent protein from a corallimor pharian, has been determined at 2.0-Angstrom resolution by multiple-wavelen gth anomalous dispersion and crystallographic refinement. Crystals of the s elenomethionine-substituted protein have space group P2(1) and contain a te tramer with 222 noncrystallographic symmetry in the asymmetric unit The ref ined model has satisfactory stereochemistry and a final crystallographic R factor of 0.162, The protein, which forms an obligatory tetramer in solutio n and in the crystal, is a squat rectangular prism comprising four protomer s whose fold is extremely similar to that of the Aequorea victoria green fl uorescent protein despite low (approximate to 23%) amino acid sequence homo logy. The monomer consists of an 11-stranded beta barrel with a coaxial hel ix The chromophores, formed from the primary sequence -Gln-Tyr-Gly- (residu es 66-68), are arranged in a approximate to 27 x 34-Angstrom rectangular ar ray in two approximately antiparallel pairs. The geometry at the cy carbon of Gln-66 (refined without stereochemical restraints) is consistent with an sp(2) hybridized center, in accord with the proposal that red fluorescence is because of an additional oxidation step that forms an acylimine extensi on to the chromophore [Gross, L. A., Baird, G. S., Hoffman, R. C, Baldridge , K. K. & Tsien, R. Y. (2000) Proc Natl. Acad. Sci. USA 87, 11990-11995]. T he carbonyl oxygen of Phe-65 is almost 90 degrees out of the plane of the c hromophore, consistent with theoretical calculations suggesting that this i s the minimum energy conformation of this moiety despite the conjugation of this group with the rest of the chromophore.