Crystallization of yeast triose phosphate isomerase from polyethylene glycol. Protein crystal formation following phase separation.

A new crystal form of yeast triose phosphate isomerase has been grown from solutions of the enzyme in polyethylene glycol (PEG) of average molecular weight 4000. The crystals are monoclinic, space group P21, with a = 61.3 A, b = 98.4 A, c = 49.7 A, /3 = 90.9”. There is one dimeric molecule per asymmetric unit. Data are observable to at least 1.3A resolution. Crystallization can be achieved after the protein is induced to “oil-out” by polyethylene glycoi precipitation. Small “oil droplets” of protein act as nucleation centers for the growth of large single crystals. Measurements of the protein and polyethylene glycol content of the two phases of the yeast triose phosphate isomerase system establish that the protein has gone entirely into the more dense phase. This phase also contains polyethylene glycol. Consideration of the behavior of this two-phase system suggests that polyethylene glycol precipitates proteins at least in part by competing for water of hydration. Since the crystals can be stored in mother liquor which does not contain either sulfate or phosphate, substrate binding experiments can be carried out in the absence of competition by these anions. Addition of even low concentrations of the transition state analogue, 2-phosphoglycolate (PGA), to the native mother liquor causes the native enzyme crystals to dissolve. Cocrystallization of yeast triose phosphate isomerase and PGA in the presence of PEG 4000 yields monoclinic crystals (space group P21, a = 74.2 A, b = 82.9 A, c = 37.6 A, /3 = 102.0’) which diffract to better than 2.0 A resolution. Addition of the substrate, dihydroxyacetone phosphate, to the native mother liquor decreases the stability of the crystals in the x-ray beam, but diffraction is observed to at least 1.8 A resolution. Consequently, the yeast triose phosphate isomerase system is suitable for a detailed mechanistic study of the reaction pathway.