139l
From Proteopedia
(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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| - | [[Image:139l.jpg|left|200px]]<br /><applet load="139l" size=" | + | [[Image:139l.jpg|left|200px]]<br /><applet load="139l" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="139l, resolution 1.7Å" /> | caption="139l, resolution 1.7Å" /> | ||
'''RAPID CRYSTALLIZATION OF T4 LYSOZYME BY INTERMOLECULAR DISULFIDE CROSSLINKING'''<br /> | '''RAPID CRYSTALLIZATION OF T4 LYSOZYME BY INTERMOLECULAR DISULFIDE CROSSLINKING'''<br /> | ||
==Overview== | ==Overview== | ||
| - | In an attempt to facilitate crystallization, engineered cysteines were | + | 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== | ==About this Structure== | ||
| - | 139L is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Enterobacteria_phage_t2 Enterobacteria phage t2] with CL and BME as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Lysozyme Lysozyme], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.17 3.2.1.17] Full crystallographic information is available from [http:// | + | 139L is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Enterobacteria_phage_t2 Enterobacteria phage t2] with <scene name='pdbligand=CL:'>CL</scene> and <scene name='pdbligand=BME:'>BME</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Lysozyme Lysozyme], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.17 3.2.1.17] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=139L OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Lysozyme]] | [[Category: Lysozyme]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Heinz, D | + | [[Category: Heinz, D W.]] |
| - | [[Category: Matthews, B | + | [[Category: Matthews, B W.]] |
[[Category: BME]] | [[Category: BME]] | ||
[[Category: CL]] | [[Category: CL]] | ||
[[Category: hydrolase(o-glycosyl)]] | [[Category: hydrolase(o-glycosyl)]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 11:38:08 2008'' |
Revision as of 09:38, 21 February 2008
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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 and 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
Page seeded by OCA on Thu Feb 21 11:38:08 2008
