139l
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(New page: 200px<br /><applet load="139l" size="450" color="white" frame="true" align="right" spinBox="true" caption="139l, resolution 1.7Å" /> '''RAPID CRYSTALLIZATION...)
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Revision as of 08:19, 20 November 2007
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RAPID CRYSTALLIZATION OF T4 LYSOZYME BY INTERMOLECULAR DISULFIDE CROSSLINKING
Overview
In an attempt to facilitate crystallization, engineered cysteines were, used to promote formation of a 'back-to-back' dimer that occurs in, different crystal forms of wild-type and mutant T4 lysozymes. The designed, double mutant, N68C/A93C, in which the surface residues Asn68 and Ala93, were replaced by cysteines, formed dimers in solution and crystallized, isomorphously to wild-type, but at a much faster rate. Overall, the mutant, structure remained very similar to wild-type despite the formation of two, intermolecular disulfide bridges. The crystals of cross-linked dimers ahd, thermal factors somewhat lower than wild-type, indicating reduced, mobility, but did not diffract to noticeably higher resolution., Introduction of the same cross-links was also used to obtain crystals in a, different space group of a T4 lysozyme mutant that could not be, crystallized previously. The results suggest that the formation of the, lysozyme dimer is a critical intermediate in the formation of more than, one crystal form and that covalent cross-linking of the intermediate, accelerates nucleation and facilitates crystal growth. The disulfide, cross-links are located on the 'back' of the molecule and formation of the, cross-linked dimer appears to leave the active sites completely, unobstructed. Nevertheless, the cross-linked dimer is completely inactive., One explanation for this behavior is that the disulfide bridges prevent, hinge-bending motion that may be required for catalysis. Another, possibility is that the formation of the dimer increases the overall bulk, of the enzyme and prevents its access to the susceptible glycosidic bonds, within the cell wall substrate.
About this Structure
139L is a Single protein structure of sequence from Enterobacteria phage t2 with CL and BME as ligands. Active as Lysozyme, with EC number 3.2.1.17 Full crystallographic information is available from OCA.
Reference
Rapid crystallization of T4 lysozyme by intermolecular disulfide cross-linking., Heinz DW, Matthews BW, Protein Eng. 1994 Mar;7(3):301-7. PMID:8177878
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