Improved Success of Sparse Matrix Protein Crystallization Screening with Heterogeneous Nucleating Agents

Crystallization is a major bottleneck in the process of macromolecular structure determination by X-ray crystallography. Successful crystallization requires the formation of nuclei and their subsequent growth to crystals of suitable size. Crystal growth generally occurs spontaneously in a supersaturated solution as a result of homogenous nucleation. However, in a typical sparse matrix screening experiment, precipitant and protein concentration are not sampled extensively, and supersaturation conditions suitable for nucleation are often missed

Plasma Dynamics

Contains table of contents for Section 2 and reports on three research projects.National Science Foundation Grant ECS 89-02990U.S. Air Force - Office of Scientific Research Grant F49620-93-1-0108U.S. Army - Harry Diamond Laboratories Contract DAAL02-92-K-0037U.S. Department of Energy Grant DE-FG02-91-ER-40648U.S. Navy - Office of Naval Research Grant N00014-90-J-4130National Aeronautics and Space Administration Grant NAGW-2048National Science Foundation Grant ECS 88-22475U.S. Department of Energy Grant DE-FG02-91-ER-54109Magnetic Fusion Science Fellowship Progra

Heterogeneous Nucleation of Protein Crystals on Fluorinated Layered Silicate

Here, we describe an improved system for protein crystallization based on heterogeneous nucleation using fluorinated layered silicate. In addition, we also investigated the mechanism of nucleation on the silicate surface. Crystallization of lysozyme using silicates with different chemical compositions indicated that fluorosilicates promoted nucleation whereas the silicates without fluorine did not. The use of synthesized saponites for lysozyme crystallization confirmed that the substitution of hydroxyl groups contained in the lamellae structure for fluorine atoms is responsible for the nucleation-inducing property of the nucleant. Crystallization of twelve proteins with a wide range of pI values revealed that the nucleation promoting effect of the saponites tended to increase with increased substitution rate. Furthermore, the saponite with the highest fluorine content promoted nucleation in all the test proteins regardless of their overall net charge. Adsorption experiments of proteins on the saponites confirmed that the density of adsorbed molecules increased according to the substitution rate, thereby explaining the heterogeneous nucleation on the silicate surface

Plasma Dynamics

Contains table of contents for Section 2 and reports on four research projects.National Science Foundation Grant ECS-89-02990U.S. Air Force - Office of Scientific Research Grant AFOSR 89-0082-CU.S. Army - Harry Diamond Laboratories Contract DAAL02-89-K-0084U.S. Army - Harry Diamond Laboratories Contract DAAL02-92-K-0037U.S. Department of Energy Contract DE-AC02-90ER-40591U.S. Navy - Office of Naval Research Grant N00014-90-J-4130Lawrence Livermore National Laboratories Subcontract B-160456National Aeronautics and Space Administration Grant NAGW-2048National Science Foundation Grant ECS-88-22475U.S. Department of Energy Grant DE-FG02-91-ER-5410

Effect of polyethylene glycol on the liquid–liquid phase transition in aqueous protein solutions

We have studied the effect of polyethylene glycol (PEG) on the liquid–liquid phase separation (LLPS) of aqueous solutions of bovine γD-crystallin (γD), a protein in the eye lens. We observe that the phase separation temperature increases with both PEG concentration and PEG molecular weight. PEG partitioning, which is the difference between the PEG concentration in the two coexisting phases, has been measured experimentally and observed to increase with PEG molecular weight. The measurements of both LLPS temperature and PEG partitioning in the ternary γD-PEG-water systems are used to successfully predict the location of the liquid–liquid phase boundary of the binary γD-water system. We show that our LLPS measurements can be also used to estimate the protein solubility as a function of the concentration of crystallizing agents. Moreover, the slope of the tie-lines and the dependence of LLPS temperature on polymer concentration provide a powerful and sensitive check of the validity of excluded volume models. Finally, we show that the increase of the LLPS temperature with PEG concentration is due to attractive protein–protein interactions