2i9k: Difference between revisions

Line 1:

[[Image:2i9k.gif|left|200px]]<br /><applet load="2i9k" size="~~450~~" color="white" frame="true" align="right" spinBox="true"

[[Image:2i9k.gif|left|200px]]<br /><applet load="2i9k" size="350" color="white" frame="true" align="right" spinBox="true"

caption="2i9k, resolution 2.65Å" />

'''Engineered Extrahelical Base Destabilization Enhances Sequence Discrimination of DNA Methyltransferase M.HhaI'''<br />

==Overview==

Improved sequence specificity of the DNA cytosine methyltransferase HhaI, was achieved by disrupting interactions at a hydrophobic interface between, the active site of the enzyme and a highly conserved flexible loop., Transient fluorescence experiments show that mutations disrupting this, interface destabilize the positioning of the extrahelical, "flipped", cytosine base within the active site. The ternary crystal structure of the, F124A M.HhaI bound to cognate DNA and the cofactor analogue, S-adenosyl-l-homocysteine shows an increase in cavity volume between the, flexible loop and the core of the enzyme. This cavity disrupts the, interface between the loop and the active site, thereby destabilizing the, extrahelical target base. The favored partitioning of the base-flipped, enzyme-DNA complex back to the base-stacked intermediate results in the, mutant enzyme discriminating better than the wild-type enzyme against, non-cognate sites. Building upon the concepts of kinetic proofreading and, our understanding of M.HhaI, we describe how a 16-fold specificity, enhancement achieved with a double mutation at the loop/active site, interface is acquired through destabilization of intermediates prior to, methyltransfer rather than disruption of direct interactions between the, enzyme and the substrate for M.HhaI.

Improved sequence specificity of the DNA cytosine methyltransferase HhaI was achieved by disrupting interactions at a hydrophobic interface between the active site of the enzyme and a highly conserved flexible loop. Transient fluorescence experiments show that mutations disrupting this interface destabilize the positioning of the extrahelical, "flipped" cytosine base within the active site. The ternary crystal structure of the F124A M.HhaI bound to cognate DNA and the cofactor analogue S-adenosyl-l-homocysteine shows an increase in cavity volume between the flexible loop and the core of the enzyme. This cavity disrupts the interface between the loop and the active site, thereby destabilizing the extrahelical target base. The favored partitioning of the base-flipped enzyme-DNA complex back to the base-stacked intermediate results in the mutant enzyme discriminating better than the wild-type enzyme against non-cognate sites. Building upon the concepts of kinetic proofreading and our understanding of M.HhaI, we describe how a 16-fold specificity enhancement achieved with a double mutation at the loop/active site interface is acquired through destabilization of intermediates prior to methyltransfer rather than disruption of direct interactions between the enzyme and the substrate for M.HhaI.

==About this Structure==

2I9K is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Haemophilus_haemolyticus Haemophilus haemolyticus] with SAH as [http://en.wikipedia.org/wiki/ligand ligand]. Full crystallographic information is available from [http://~~ispc~~.weizmann.ac.il/oca-bin/ocashort?id=2I9K OCA].

2I9K is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Haemophilus_haemolyticus Haemophilus haemolyticus] with <scene name='pdbligand=SAH:'>SAH</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2I9K OCA].

==Reference==

Line 13:

[[Category: Haemophilus haemolyticus]]

[[Category: Single protein]]

[[Category: Perona, J.J.]]

[[Category: Perona, J J.]]

[[Category: Reich, N.O.]]

[[Category: Reich, N O.]]

[[Category: Rios, S.De.Los.]]

[[Category: Rios, S De Los.]]

[[Category: Shieh, F.K.]]

[[Category: Shieh, F K.]]

[[Category: Youngblood, B.]]

[[Category: SAH]]

[[Category: phe124ala mutation in m.hhai]]

[[Category: phe124ala mutation in m hhai]]

''Page seeded by [http://~~ispc~~.weizmann.ac.il/oca OCA ] on ~~Wed Nov~~ 21 12:11:~~56 2007~~''

''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 17:50:23 2008''

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@@ Line 1: / Line 1: @@
-[[Image:2i9k.gif|left|200px]]<br /><applet load="2i9k" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:2i9k.gif|left|200px]]<br /><applet load="2i9k" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="2i9k, resolution 2.65&Aring;" />
 '''Engineered Extrahelical Base Destabilization Enhances Sequence Discrimination of DNA Methyltransferase M.HhaI'''<br />
 ==Overview==
-Improved sequence specificity of the DNA cytosine methyltransferase HhaI, was achieved by disrupting interactions at a hydrophobic interface between, the active site of the enzyme and a highly conserved flexible loop., Transient fluorescence experiments show that mutations disrupting this, interface destabilize the positioning of the extrahelical, "flipped", cytosine base within the active site. The ternary crystal structure of the, F124A M.HhaI bound to cognate DNA and the cofactor analogue, S-adenosyl-l-homocysteine shows an increase in cavity volume between the, flexible loop and the core of the enzyme. This cavity disrupts the, interface between the loop and the active site, thereby destabilizing the, extrahelical target base. The favored partitioning of the base-flipped, enzyme-DNA complex back to the base-stacked intermediate results in the, mutant enzyme discriminating better than the wild-type enzyme against, non-cognate sites. Building upon the concepts of kinetic proofreading and, our understanding of M.HhaI, we describe how a 16-fold specificity, enhancement achieved with a double mutation at the loop/active site, interface is acquired through destabilization of intermediates prior to, methyltransfer rather than disruption of direct interactions between the, enzyme and the substrate for M.HhaI.
+Improved sequence specificity of the DNA cytosine methyltransferase HhaI was achieved by disrupting interactions at a hydrophobic interface between the active site of the enzyme and a highly conserved flexible loop. Transient fluorescence experiments show that mutations disrupting this interface destabilize the positioning of the extrahelical, "flipped" cytosine base within the active site. The ternary crystal structure of the F124A M.HhaI bound to cognate DNA and the cofactor analogue S-adenosyl-l-homocysteine shows an increase in cavity volume between the flexible loop and the core of the enzyme. This cavity disrupts the interface between the loop and the active site, thereby destabilizing the extrahelical target base. The favored partitioning of the base-flipped enzyme-DNA complex back to the base-stacked intermediate results in the mutant enzyme discriminating better than the wild-type enzyme against non-cognate sites. Building upon the concepts of kinetic proofreading and our understanding of M.HhaI, we describe how a 16-fold specificity enhancement achieved with a double mutation at the loop/active site interface is acquired through destabilization of intermediates prior to methyltransfer rather than disruption of direct interactions between the enzyme and the substrate for M.HhaI.
 ==About this Structure==
-I9K is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Haemophilus_haemolyticus Haemophilus haemolyticus] with SAH as [http://en.wikipedia.org/wiki/ligand ligand]. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=2I9K OCA].
+I9K is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Haemophilus_haemolyticus Haemophilus haemolyticus] with <scene name='pdbligand=SAH:'>SAH</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2I9K OCA].
 ==Reference==
@@ Line 13: / Line 13: @@
 [[Category: Haemophilus haemolyticus]]
 [[Category: Single protein]]
-[[Category: Perona, J.J.]]
+[[Category: Perona, J J.]]
-[[Category: Reich, N.O.]]
+[[Category: Reich, N O.]]
-[[Category: Rios, S.De.Los.]]
+[[Category: Rios, S De Los.]]
-[[Category: Shieh, F.K.]]
+[[Category: Shieh, F K.]]
 [[Category: Youngblood, B.]]
 [[Category: SAH]]
-[[Category: phe124ala mutation in m.hhai]]
+[[Category: phe124ala mutation in m hhai]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Wed Nov 21 12:11:56 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 17:50:23 2008''