title: Proline: Mother Nature's cryoprotectant applied to protein crystallography.
abstract: L-Proline is one of Mother Nature's cryoprotectants. Plants and yeast accumulate proline under freeze-induced stress and the use of proline in the cryopreservation of biological samples is well established. Here, it is shown that L-proline is also a useful cryoprotectant for protein crystallography. Proline was used to prepare crystals of lysozyme, xylose isomerase, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase for low-temperature data collection. The crystallization solutions in these test cases included the commonly used precipitants ammonium sulfate, sodium chloride and polyethylene glycol and spanned the pH range 4.6-8.5. Thus, proline is compatible with typical protein-crystallization formulations. The proline concentration needed for cryoprotection of these crystals is in the range 2.0-3.0 M. Complete data sets were collected from the proline-protected crystals. Proline performed as well as traditional cryoprotectants based on the diffraction resolution and data-quality statistics. The structures were refined to assess the binding of proline to these proteins. As observed with traditional cryoprotectants such as glycerol and ethylene glycol, the electron-density maps clearly showed the presence of proline molecules bound to the protein. In two cases, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase, proline binds in the active site. It is concluded that L-proline is an effective cryoprotectant for protein crystallography.
authors.affiliation: Department of Chemistry, University of Missouri-Columbia, Columbia, MO 65211, USA.
authors.forename: Travis A
authors.initials: TA
authors.lastname: Pemberton
authors.forename: Brady R
authors.initials: BR
authors.lastname: Still
authors.forename: Emily M
authors.initials: EM
authors.lastname: Christensen
authors.forename: Harkewal
authors.initials: H
authors.lastname: Singh
authors.forename: Dhiraj
authors.initials: D
authors.lastname: Srivastava
authors.forename: John J
authors.initials: JJ
authors.lastname: Tanner
mesh_terms: D050842:1-Pyrroline-5-Carboxylate Dehydrogenase / Q000737:chemistry; D000135:Acid Phosphatase / Q000737:chemistry; D019747:Aldose-Ketose Isomerases / Q000737:chemistry; D000818:Animals; D002645:Chickens; D003451:Cryoprotective Agents / Q000737:chemistry* / Q000494:pharmacology; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods*; D004529:Egg White; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009113:Muramidase / Q000737:chemistry; D011392:Proline / Q000737:chemistry*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry; D013696:Temperature
chemical_list: D003451:Cryoprotective Agents; D011506:Proteins; D006639:Histidine; D011392:Proline; D050842:1-Pyrroline-5-Carboxylate Dehydrogenase; D000135:Acid Phosphatase; D009113:Muramidase; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase