title: Thermofluor-based high-throughput stability optimization of proteins for structural studies.
abstract: Production of proteins well suited for structural studies is inherently difficult and time-consuming. Protein sample homogeneity, stability, and solubility are strongly correlated with the proteins' probability of yielding crystals, and optimization of these properties will improve success rates of crystallization. In the current study, we applied the thermofluor method as a high-throughput approach for identifying optimal protein formulation for crystallization. The method also allowed optimal stabilizing buffer compositions to be rapidly identified for each protein. Furthermore, the method allowed the identification of potential ligands, physiological or non-physiological, that can be used in subsequent crystallization trials. For this study, the thermally induced melting points were determined in different buffers as well as with additives for a total of 25 Escherichia coli proteins. Crystallization trials were set up together with stabilizing and destabilizing additives identified using thermofluor screening. A twofold increase in the number of crystallization leads was observed when the proteins were cocrystallized with stabilizing additives as compared with experiments without these additives. This suggests that thermofluor constitutes an efficient generic high-throughput method for identification of protein properties predictive of crystallizability.
authors.affiliation: Department of Biochemistry and Biophysics, Stockholm University, Stockholm SE-109 51, Sweden.
authors.forename: Ulrika B
authors.initials: UB
authors.lastname: Ericsson
authors.forename: B Martin
authors.initials: BM
authors.lastname: Hallberg
authors.forename: George T
authors.initials: GT
authors.lastname: Detitta
authors.forename: Niek
authors.initials: N
authors.lastname: Dekker
authors.forename: Pär
authors.initials: P
authors.lastname: Nordlund
mesh_terms: D002021:Buffers; D003460:Crystallization; D008958:Models, Molecular; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D013696:Temperature; D013816:Thermodynamics
chemical_list: D002021:Buffers; D011506:Proteins