Optimum Solubility Screen To optimize buffer conditions for homogeneity and crystallization of proteins Jarmila Jancarik, Ramona Pufan, Connie Hong and Rosalind Kim Lawrence Berkeley National Laboratory PPCW, March 29-31, 2004 The work described here was supported by the National Institutes of Health GM 62412

Step 1 Optimium Solubility Screen Aggregated protein in purification buffer 1 µl Buffer 1 µl Protein Set up protein using 24-well Linbro plates, use 0.5 ml buffer (24) in reservoir; 1 µl buffer + 1 µl protein on the cover slip (vapor diffusion method) Incubate overnight over the same buffer buffer Perform Dynamic Light Scattering (DLS) on clear drops, pick best condition.

List of optimization buffers Linbro Plate List of optimization buffers Version 1

Additive Screen Step 2 Test additives using best buffer: 25, 50, 100 mM NaCl 5 and 10% glycerol 2 mM CHAPS (CMC=6 to 10 mM) 0.1%, 1% Octylglucoside (CMC=0.53%) 0.1%, 1% Dodecyl Maltoside (CMC=0.0087%) 30mM TCEP, 5mM DTT,10 mM β-mercaptoethanol Two hour incubation Perform DLS Pick best additive, exchange protein into optimal buffer/additive Set up Crystal Screens

DLS in Different Buffers 1371B in Buffer #16 100 mM Na K Phosphate, pH 7.0 1371B in Buffer #21 100 mM Tris, pH 8.5

1371B 1371B in 100 mM Tris, pH 8.5 1% Octylglucoside Crystals of 1371B Structure solved

Results * Radius in nanometers, % polydispersity **Crystals appeared in Optimium Screen

Summary This assay provides an opportunity to find an optimum buffer/additive for crystallization of a given protein. Screen is fast and requires a small amount of protein. Out of 14 targets, protein monodispersity improved greatly for 11 targets and 9 of these proteins crystallized. This screen is being implemented into a high throughput protocol.