An Extensive Study of Protein Phase Diagram Modification: Increasing Macromolecular Crystallizability by Temperature Screening

A new parameter relative crystallizability for protein crystallization has been proposed, and its relationship with protein solubility and crystallization success has been studied (Zhu et al. J. Struct. Biol. 2006, 154, 297). Here we further construct the phase diagrams of a larger number of proteins, study the phase modification as a function of temperature, and establish the relationship between the nucleation zone area (S-N) and crystallization success. The phase diagrams of 10 proteins were constructed and their SN were compared, demonstrating that temperature modifies the protein nucleation zone. Such modification can significantly enlarge the SN and increase protein crystallizability. For example, the SN of ribonuclease S and trypsin increases by 2.4- and 1.6-fold when the temperature moves to 277 K from 295 K, while at the same time the crystallization hits increase from 20.8% to 42.9% and 12.5% to 25%, respectively. S-N of chymotrypsinogen A and concanavalin A increases by 1.6- and 1.7-fold (277 to 295 K), while the hits increase from 37.5% to 54.2% and 43.3% to 73.4%, respectively. Such an excellent agreement strongly supports the validity of protein relative crystallizability, and crystallization screening at several temperatures can significantly increase the success for most proteins. A new protein epididymal-specific lipocalin was crystallized by varying temperature, yielding quickly the first crystals, and complete data sets have been collected at 1.97 angstrom.