1p6y: Difference between revisions

Revision as of 15:25, 21 February 2008

T4 LYSOZYME CORE REPACKING MUTANT M120Y/TA

OverviewOverview

Automated protein redesign, as implemented in the program ORBIT, was used to redesign the core of phage T4 lysozyme. A total of 26 buried or partially buried sites in the C-terminal domain were allowed to vary both their sequence and side-chain conformation while the backbone and non-selected side-chains remained fixed. A variant with seven substitutions ("Core-7") was identified as having the most favorable energy. The redesign experiment was repeated with a penalty for the presence of methionine residues. In this case the redesigned protein ("Core-10") had ten amino acid changes. The two designed proteins, as well as the constituent single mutants, and several single-site revertants were over-expressed in Escherichia coli, purified, and subjected to crystallographic and thermal analyses. The thermodynamic and structural data show that some repacking was achieved although neither redesigned protein was more stable than the wild-type protein. The use of the methionine penalty was shown to be effective. Several of the side-chain rotamers in the predicted structure of Core-10 differ from those observed. Rather than changing to new rotamers predicted by the design process, side-chains tend to maintain conformations similar to those seen in the native molecule. In contrast, parts of the backbone change by up to 2.8A relative to both the designed structure and wild-type.Water molecules that are present within the lysozyme molecule were removed during the design process. In the redesigned protein the resultant cavities were, to some degree, re-occupied by side-chain atoms. In the observed structure, however, water molecules were still bound at or near their original sites. This suggests that it may be preferable to leave such water molecules in place during the design procedure. The results emphasize the specificity of the packing that occurs within the core of a typical protein. While point substitutions within the core are tolerated they almost always result in a loss of stability. Likewise, combinations of substitutions may also be tolerated but usually destabilize the protein. Experience with T4 lysozyme suggests that a general core repacking methodology with retention or enhancement of stability may be difficult to achieve without provision for shifts in the backbone.

About this StructureAbout this Structure

1P6Y is a Single protein structure of sequence from Bacteriophage t4 with , , and as ligands. Active as Lysozyme, with EC number 3.2.1.17 Full crystallographic information is available from OCA.

ReferenceReference

Repacking the Core of T4 lysozyme by automated design., Mooers BH, Datta D, Baase WA, Zollars ES, Mayo SL, Matthews BW, J Mol Biol. 2003 Sep 19;332(3):741-56. PMID:12963380

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-[[Image:1p6y.jpg|left|200px]]<br /><applet load="1p6y" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1p6y.jpg|left|200px]]<br /><applet load="1p6y" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1p6y, resolution 1.54&Aring;" />
 '''T4 LYSOZYME CORE REPACKING MUTANT M120Y/TA'''<br />
 ==Overview==
-Automated protein redesign, as implemented in the program ORBIT, was used, to redesign the core of phage T4 lysozyme. A total of 26 buried or, partially buried sites in the C-terminal domain were allowed to vary both, their sequence and side-chain conformation while the backbone and, non-selected side-chains remained fixed. A variant with seven, substitutions ("Core-7") was identified as having the most favorable, energy. The redesign experiment was repeated with a penalty for the, presence of methionine residues. In this case the redesigned protein, ("Core-10") had ten amino acid changes. The two designed proteins, as well, as the constituent single mutants, and several single-site revertants were, over-expressed in Escherichia coli, purified, and subjected to, crystallographic and thermal analyses. The thermodynamic and structural, data show that some repacking was achieved although neither redesigned, protein was more stable than the wild-type protein. The use of the, methionine penalty was shown to be effective. Several of the side-chain, rotamers in the predicted structure of Core-10 differ from those observed., Rather than changing to new rotamers predicted by the design process, side-chains tend to maintain conformations similar to those seen in the, native molecule. In contrast, parts of the backbone change by up to 2.8A, relative to both the designed structure and wild-type.Water molecules that, are present within the lysozyme molecule were removed during the design, process. In the redesigned protein the resultant cavities were, to some, degree, re-occupied by side-chain atoms. In the observed structure, however, water molecules were still bound at or near their original sites., This suggests that it may be preferable to leave such water molecules in, place during the design procedure. The results emphasize the specificity, of the packing that occurs within the core of a typical protein. While, point substitutions within the core are tolerated they almost always, result in a loss of stability. Likewise, combinations of substitutions may, also be tolerated but usually destabilize the protein. Experience with T4, lysozyme suggests that a general core repacking methodology with retention, or enhancement of stability may be difficult to achieve without provision, for shifts in the backbone.
+Automated protein redesign, as implemented in the program ORBIT, was used to redesign the core of phage T4 lysozyme. A total of 26 buried or partially buried sites in the C-terminal domain were allowed to vary both their sequence and side-chain conformation while the backbone and non-selected side-chains remained fixed. A variant with seven substitutions ("Core-7") was identified as having the most favorable energy. The redesign experiment was repeated with a penalty for the presence of methionine residues. In this case the redesigned protein ("Core-10") had ten amino acid changes. The two designed proteins, as well as the constituent single mutants, and several single-site revertants were over-expressed in Escherichia coli, purified, and subjected to crystallographic and thermal analyses. The thermodynamic and structural data show that some repacking was achieved although neither redesigned protein was more stable than the wild-type protein. The use of the methionine penalty was shown to be effective. Several of the side-chain rotamers in the predicted structure of Core-10 differ from those observed. Rather than changing to new rotamers predicted by the design process, side-chains tend to maintain conformations similar to those seen in the native molecule. In contrast, parts of the backbone change by up to 2.8A relative to both the designed structure and wild-type.Water molecules that are present within the lysozyme molecule were removed during the design process. In the redesigned protein the resultant cavities were, to some degree, re-occupied by side-chain atoms. In the observed structure, however, water molecules were still bound at or near their original sites. This suggests that it may be preferable to leave such water molecules in place during the design procedure. The results emphasize the specificity of the packing that occurs within the core of a typical protein. While point substitutions within the core are tolerated they almost always result in a loss of stability. Likewise, combinations of substitutions may also be tolerated but usually destabilize the protein. Experience with T4 lysozyme suggests that a general core repacking methodology with retention or enhancement of stability may be difficult to achieve without provision for shifts in the backbone.
 ==About this Structure==
-P6Y is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bacteriophage_t4 Bacteriophage t4] with PO4, K, CL and HED 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://ispc.weizmann.ac.il/oca-bin/ocashort?id=1P6Y OCA].
+P6Y is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bacteriophage_t4 Bacteriophage t4] with <scene name='pdbligand=PO4:'>PO4</scene>, <scene name='pdbligand=K:'>K</scene>, <scene name='pdbligand=CL:'>CL</scene> and <scene name='pdbligand=HED:'>HED</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=1P6Y OCA].
 ==Reference==
@@ Line 14: / Line 14: @@
 [[Category: Lysozyme]]
 [[Category: Single protein]]
-[[Category: Baase, W.A.]]
+[[Category: Baase, W A.]]
 [[Category: Datta, D.]]
-[[Category: Matthews, B.W.]]
+[[Category: Matthews, B W.]]
-[[Category: Mayo, S.L.]]
+[[Category: Mayo, S L.]]
-[[Category: Mooers, B.H.]]
+[[Category: Mooers, B H.]]
-[[Category: Zollars, E.S.]]
+[[Category: Zollars, E S.]]
 [[Category: CL]]
 [[Category: HED]]
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 [[Category: t4 lysozyme]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 23:32:03 2007''
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