Pentaerythritol propoxylate: a new crystallization agent and cryoprotectant induces crystal growth of 2-methylcitrate dehydratase.
In the search for macromolecular crystallization conditions, the precipitant is probably the most important variable, such that when problematic crystals are encountered there is always the question of whether an alternative precipitant might resolve the problem. During an effort to obtain high-quality crystals of several problematic proteins, two new agents, pentaerythritol propoxylate and pentaerythritol ethoxylate, yielded well ordered quality crystals where more traditional precipitants were unsuccessful. Pentaerythritol propoxylate and pentaerythritol ethoxylate contain a pentaerythritol backbone to which organic polymers are bound, forming a branched polymer. As such, they are larger than small organic precipitants such as low molecular-weight alcohols or 2-methyl-2,4-pentanediol, but behave differently to polyethylene glycols. These compounds have been used to crystallize an enzyme encoded by the Salmonella enterica prpD gene that catalyzes the dehydration of 2-methylcitrate to form 2-methyl-cis-aconitate. While the PrpD protein has crystallized readily under a number of conditions, the resultant crystals were unsuitable for a crystal structure determination. The new crystals obtained with 25-40% pentaerythritol propoxylate belong to the orthorhombic space group C222(1), with unit-cell parameters a = 73.2, b = 216.4, c = 214.3 A, and diffract beyond 2.0 A with synchrotron radiation. A further benefit of this precipitant for crystallization is its ability to function as a cryoprotectant, allowing the crystals to be transferred directly from the mother liquor to the nitrogen stream at 113 K.
Gulick
Andrew M
AM
Department of Biochemistry, University of Wisconsin, 433 Babcock Drive, Madison, WI 53706, USA.
Horswill
Alexander R
AR
Thoden
James B
JB
Escalante-Semerena
Jorge C
JC
Rayment
Ivan
I
D000154:Aconitate Hydratase / Q000737:chemistry* / Q000235:genetics; D003451:Cryoprotective Agents / Q000737:chemistry*; D003460:Crystallization; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D011108:Polymers / Q000737:chemistry*; D011487:Protein Conformation; D019779:Salmonella enterica / Q000737:chemistry / Q000235:genetics; D013402:Sugar Alcohols / Q000737:chemistry*
D003451:Cryoprotective Agents; D011108:Polymers; D013402:Sugar Alcohols; C455288:pentaerythritol propoxylate; D000154:Aconitate Hydratase