5.2. Crystallization of yeast Δ3-Δ2-enoyl-CoA isomerase (II, IV)
The initial crystallization conditions for yeast enoyl-CoA isomerase were searched for by using Hampton Screen and Hampton Screen II (Hampton Research). The most promising conditions were optimized with respect to pH and the precipitant concentration. Two unliganded crystal forms were obtained at room temperature. Crystals of tetragonal shape grew in 0.1 M sodium acetate pH 4.8, 2.2 M (NH4)2SO4 when 1 mg/ml protein solution was used (Fig. 10A). The crystals had maximum dimensions of 0.3 x 0.2 x 0.2 mm3, and they diffracted to about 3 Å resolution when the rotating anode X-ray source was used.
The crystal belonged to a primitive tetragonal space group and the unit cell dimensions were a=b=118.1 Å, c=222.3 Å, α=β =γ = 90°. This crystal form was not analyzed further. The other crystal form was obtained in 0.1 MES pH 5.5, 5 % 1,4-dioxane, 1.4 M (NH4)2SO4 using a protein concentration of 2.3 mg/ml and 10 mM N-octyl-β -D-glucoside as an additive (Fig 10B). The crystals had hexagonal shape and they belonged to the space group P6322 with unit cell dimensions of a=b=116.1 Å, c=123.3 Å, α=β = 90°, γ =120° and one monomer per asymmetric unit. The diffraction limit of the hexagonal crystals was better than 3 Å when using the home X-ray source and 2.15 Å when synchrotron radiation was used at DESY, Hamburg. These crystals were easy to obtain and could be frozen in the presence of 20 % ethylene glycol, in addition to which they had higher symmetry than the tetragonal crystals. Thus, the hexagonal unliganded crystal form was chosen to be used in the structure determination of the yeast enoyl-CoA isomerase.
The first attempts to obtain crystals liganded with various fatty acyl-CoA esters were made by soaking unliganded hexagonal crystals in solutions containing fatty acyl-CoAs. This did not lead to ligand binding, however, possibly because of the low pH of the hexagonal crystals. The next step was to use co-crystallization. Prior to the crystallization experiments, the enoyl-CoA isomerase was concentrated to 2.5 mg/ml and octanoyl-CoA was added to the solution in a concentration of 2 mM. Octanoyl-CoA is an inhibitor of the enoyl-CoA isomerase that can bind to the active site but cannot be used as a substrate. In order to facilitate ligand binding, crystallization conditions with neutral or slightly basic pH were screened and further optimized. The best defined crystals were obtained in 0.1 M TEA, pH 6.5-7.0, 1.2-1.4 M sodium citrate (Fig. 10C). The crystals were quite small, about 0.15 mm in their longest dimension, and diffracted only to 3 Å resolution using synchrotron radiation at Max-Lab in Lund. Prior to data collection, the crystals were frozen using 20 % glycerol as cryo protectant. The crystals belonged to the primitive tetragonal space group such as P422 and had a trimer in the asymmetric unit. The unit cell dimensions were a=b=116.7 Å, c=216.8 Å, α=β =γ = 90°. Another octanoyl-CoA-liganded, hexagonal crystal form was obtained in 0.1 M sodium acetate pH 5.1, 1.7 M (NH4)2SO4, but because of its low crystallization pH, it was not used in the structure determination.