Abstract
Crystallization is a limiting step in macromolecular crystallography. In cases where proteins are particularly recalcitrant to crystallization efforts, mutational modification of surface properties is a possible remedy. We previously suggested that targeted replacement of clusters of residues with high‐conformational entropy (lysines, glutamates and/or glutamines) with alanines leads to formation of epitopes that are capable of mediating crystal contacts (1). This method allowed for successful crystallization and structure determination of a number of novel proteins and to grow crystals diffracting to significantly higher resolution than those of the wild‐type form. However, it has not been conclusively demonstrated if alanine constitutes the best choice for replacement of high‐entropy residues. Here we present a systematic study of the replacement of eight Lys/Glu‐rich patches in a model molecule of RhoGDI with Ser, Thr, His and Tyr residues. Our data show that tyrosines may be particularly useful to generate crystal contacts, as RhoGDI mutants with Tyr‐rich patches give numerous hits in crystallization screens. Several crystal structures of surface mutants will be presented, showing how the mutated surface patches mediate crystal contacts through either homotypic (symmetric) or heterotypic (head‐to‐tail) intermolecular interactions.
The ISFI is supported by the NIGMS Protein Structure Initiative.