Rapid crystallization of T4 lysozyme by intermolecular disulfide cross-linking

In an attempt to facilitate crystallization, engineered cysteineswere used to promote formation of a ‘back–to–back’dimer that occurs in different crystal forms of wild–typeand mutant T4 lysozymes. The designed double mutant, N68C/A93C,in which the surface residues Asn68 and Ala93 were replacedby cysteines, formed dimers in solution and crystallized isomorphouslyto wild–type, but at a much faster rate. Overall, themutant structure remained very similar to wildtype despite theformation of two intermolecular disulfide bridges. The crystalsof cross–linked dimers had thermal factors somewhat lowerthan wild–type, indicating reduced mobility, but did notdiffract to noticeably higher resolution. Introduction of thesame cross-links was also used to obtain crystals in a differentspace group of a T4 lysozyme mutant that could not be crystallizedpreviously. The results suggest that the formation of the lysozymedimer is a critical intermediate in the formation of more thanone crystal form and that covalent cross–Unking of theintermediate accelerates nucleation and facilitates crystalgrowth. The disulfide crosslinks are located on the ‘back’of the molecule and formation of the cross–linked dimerappears to leave the active sites completely unobstructed. Nevertheless,the cross–linked dimer is completely inactive. One explanationfor this behavior is that the disulfide bridges prevent hinge-bendingmotion that may be required for catalysis. Another possibilityis that the formation of the dimer increases the overall bulkof the enzyme and prevents its access to the susceptible glycosidkbonds within the cell wall substrate