Crystallization of macromolecular complexes: stoichiometric variation screening

Theoretically a crystal may contain both complexed and uncomplexed molecule s simultaneously in the same lattice. Since we seldom screen for such possi bilities, such occurrences are only rarely reported. Here we propose that s toichiometry should be one of the parameters to be screened in the crystall ization of macromolecular complexes. By allowing for non-biologically signi ficant stoichiometries, we may increase the chances of crystallizing a macr omolecular complex and of selecting arrangements which crystallize better o r yield more ordered crystals. Although biological forces tend to be strong er than lattice-building interactions, in the crystal the latter will domin ate numerically. By allowing for a varied stoichiometry we permit a wider s election of lattice-building contacts and increase the probability of cryst allization. From these theoretical considerations we have developed methodo logy compatible with classical solubility screening and other well-establis hed crystallization principles. We discuss this technique, stoichiometric v ariation screening (SVS), as part of a multicomponent system for the enhanc ement of crystallization of macromolecular complexes. We present this techn ique as an extension of reverse screening and illustrate the complementarit y in the methodology. We present two examples of the use of SVS: the comple xes between an immunoglobulin Fab fragment and two bacterial proteins, name ly the D domain of protein A from Staphylococcus aureus (SpA) and a single domain of protein L from Peptostreptococcus magnus (PpL). In the first exam ple there are 3 Fab molecules and only 2 SpA D domains (domD) (2 complexed and I unliganded Fab), in the second 2 Fabs and only I PpL domain (I comple xed and I unliganded Fab). SVS has the added and unique advantage that in t he same crystal we have information on both the unliganded and complexed st ates under precisely identical conditions: one structure, two answers. Toge ther with a combinatorial method for complex crystallization based on immun oglobulin-binding domains, it may enhance the probability of crystallizatio n by well over a factor of ten. (C) 2001 Elsevier Science B.V. All rights r eserved.