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

Abstract

The crystal structure of DsRed, a red fluorescent protein from a corallimorpharian, has been determined at 2.0-A resolution by multiple-wavelength anomalous dispersion and crystallographic refinement. Crystals of the selenomethionine-substituted protein have space group P2(1) and contain a tetramer with 222 noncrystallographic symmetry in the asymmetric unit. The refined model has satisfactory stereochemistry and a final crystallographic R factor of 0.162. The protein, which forms an obligatory tetramer in solution and in the crystal, is a squat rectangular prism comprising four protomers whose fold is extremely similar to that of the Aequorea victoria green fluorescent protein despite low ( approximately 23%) amino acid sequence homology. The monomer consists of an 11-stranded beta barrel with a coaxial helix. The chromophores, formed from the primary sequence -Gln-Tyr-Gly- (residues 66-68), are arranged in a approximately 27 x 34-A rectangular array in two approximately antiparallel pairs. The geometry at the alpha 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 extension 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]. The carbonyl oxygen of Phe-65 is almost 90 degrees out of the plane of the chromophore, consistent with theoretical calculations suggesting that this is the minimum energy conformation of this moiety despite the conjugation of this group with the rest of the chromophore.

Scheme 1

Figure 1

Stereo view of superposition of the α-carbon backbones of avGFP (filled bonds) and DsRed (open bonds), showing the DsRed chromophore in ball-and-stick representation.

Figure 2

Ribbon diagram of the DsRed tetramer, produced by molscript (28). Monomers are labeled with uppercase A–D, and the carboxy termini are labeled with lowercase a–d. The amino termini are obscured in this view. Note the antenna-like array of the chromophores, antiparallel in pairs.

Figure 3

(A) A portion of the experimental multiwavelength anomalous dispersion-phased electron density map at 2.0-Å resolution, contoured at 1.0 standard deviation. The refined model of the chromophore and some surrounding side chains are superimposed. (B) Ball-and-stick diagram of the DsRed chromophore and environment. Some important salt bridges and/or hydrogen bonds are indicated by dashed lines. (C) Schematic diagram of the chromophore environment showing salt bridges and/or hydrogen bonds (dashed), labeled with approximate lengths in angstroms. Charged residues in the vicinity of the chromophore are colored, and a proposed catalytic water molecule is indicated by W* (see text).

Scheme 2