Alternative Protein Crystallization Technique: Cross-Influence Procedure (CIP)

Abstract

In general, the crystallization of proteins is a very complex process. Experiences of many scientists point out that majority of proteins is difficult to crystallize and even if a protein tends to crystallize relatively easily there are many parameters that must be taken into account. There are multiple reasons that point out the difficulty of protein crystal growth. Apparently, protein molecules are very complex (large, flexible molecules often composed of several subunits), relatively chemically and physically unstable (unfolding, hydration requirements, temperature sensitivity) and they have dynamic properties. If the solution changes, the molecule properties (e.g. conformation, charge and size) will change too. Furthermore, every macromolecule is unique in its physical and chemical properties since every amino acid sequence produces a unique three-dimensional structure having distinctive surface characteristics. Thus, conditions applied for one protein can only marginally apply to others (Giacovazzo, 2002; Lattman, 2008). Therefore, finding of successful crystallization conditions for a particular protein remains a highly empirical process. During optimization a variable set of parameters is screened to determine appropriate conditions for nucleation and growth of single crystals suitable for Xray diffraction analysis. In parallel to modern high-throughput approaches used in the protein crystallization, in recent years we performed basic research on physico-chemical properties and molecular interactions influencing crystal growth. Empirically, we have explored another tool useful for optimization strategy that was first described by Tomcova and Kuta Smatanova (2007). A new crystallization procedure modifying protein crystal morphology, internal packing and influencing crystal growth was tested particularly. For the first time the metal ion salts were added simultaneously to the protein drop and even to neighboring drops to allow a cross-influence effect of additives during crystallization experiment. The presence of metal ions significantly influences the crystal growth, as the modification of crystal morphology and internal packing were observed. This newly discovered cross-crystallization method (Tomcova & Kuta Smatanova, 2007; Tomcova et al., 2006) was called Cross-Influence Procedure (CIP).