High-resolution diffracting crystals of intrinsically active p38alpha MAP kinase: a case study for low-throughput approaches.
p38 MAP kinases are central signalling molecules that mediate cellular responses to numerous environmental conditions and signalling molecules. Their proper function is required for many processes, including stress response, apoptosis, differentiation, growth and even learning and memory. Abnormal activity of p38 MAP kinases is associated with the aetiology of many diseases, making understanding their activation mechanisms highly critical. In this respect, mechanistic insights may be derived from structural studies of recently developed intrinsically active p38alpha mutants. Unlike wild-type p38alpha, which routinely crystallized, the active mutants caused severe difficulties during the crystallization process. The main hindrance was found to be protein heterogeneity, which was meticulously resolved by genetically modifying the recombinant protein and optimizing the expression and purification protocols. The success in obtaining crystallizable proteins strongly emphasizes that in certain cases, high-throughput techniques (crystallization robots) together with low-throughput approaches, with careful monitoring and analysis of the results, are essential.
Diskin
Ron
R
The Wolfson Centre for Applied Structural Biology, The Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Givat Ram, Jerusalem 91904, Israel.
Engelberg
David
D
Livnah
Oded
O
D003460:Crystallization; D018360:Crystallography, X-Ray; D048308:Mitogen-Activated Protein Kinase 14 / Q000737:chemistry* / Q000235:genetics; D009154:Mutation
D048308:Mitogen-Activated Protein Kinase 14