1lav: Difference between revisions
New page: left|200px<br /><applet load="1lav" size="450" color="white" frame="true" align="right" spinBox="true" caption="1lav, resolution 1.8Å" /> '''STABILIZATION OF ESCH... |
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[[Image:1lav.gif|left|200px]]<br /><applet load="1lav" size=" | [[Image:1lav.gif|left|200px]]<br /><applet load="1lav" size="350" color="white" frame="true" align="right" spinBox="true" | ||
caption="1lav, resolution 1.8Å" /> | caption="1lav, resolution 1.8Å" /> | ||
'''STABILIZATION OF ESCHERICHIA COLI RIBONUCLEASE HI BY CAVITY-FILLING MUTATIONS WITHIN A HYDROPHOBIC CORE'''<br /> | '''STABILIZATION OF ESCHERICHIA COLI RIBONUCLEASE HI BY CAVITY-FILLING MUTATIONS WITHIN A HYDROPHOBIC CORE'''<br /> | ||
==Overview== | ==Overview== | ||
The crystal structure of Escherichia coli ribonuclease HI has a cavity | The crystal structure of Escherichia coli ribonuclease HI has a cavity near Val-74 within the protein core. In order to fill the cavity space, we constructed two mutant proteins, V74L and V74I, in which Val-74 was replaced with either Leu or Ile, respectively. The mutant proteins are stabilized, as revealed by a 2.1-3.7 degrees C increase in the Tm values, as compared to the wild-type protein at pH values of 3.0 and 5.5. The mutant protein V74A, in which Val-74 is replaced with Ala, was also constructed to analyze the reverse effect. The stability of V74A decreases by 7.6 degrees C at pH 3.0 and 12.7 degrees C at pH 5.5 in Tm as compared to those values for the wild-type protein. None of the three mutations significantly affect the enzymatic activity. The crystal structures of V74L and V74I, determined at 1.8-A resolution, are almost identical to that of the wild-type protein, except for the mutation site. In the two mutant proteins, calculation by the Voronoi procedure shows that the cavity volumes around the individual mutation sites are remarkably reduced as compared to that in the wild-type protein. These results indicate that the introduction of a methylene group into the cavity, without causing steric clash, contributes to an increase in the hydrophobic interaction within the protein core and thereby enhances protein stability. We also discuss the role of the Leu side chain, which can assume many different local conformations on a helix without sacrificing thermostability. | ||
==About this Structure== | ==About this Structure== | ||
1LAV is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli]. Active as [http://en.wikipedia.org/wiki/Ribonuclease_H Ribonuclease H], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.26.4 3.1.26.4] Full crystallographic information is available from [http:// | 1LAV is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli]. Active as [http://en.wikipedia.org/wiki/Ribonuclease_H Ribonuclease H], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.26.4 3.1.26.4] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1LAV OCA]. | ||
==Reference== | ==Reference== | ||
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[[Category: hydrolase]] | [[Category: hydrolase]] | ||
''Page seeded by [http:// | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:43:13 2008'' | ||
Revision as of 14:43, 21 February 2008
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STABILIZATION OF ESCHERICHIA COLI RIBONUCLEASE HI BY CAVITY-FILLING MUTATIONS WITHIN A HYDROPHOBIC CORE
OverviewOverview
The crystal structure of Escherichia coli ribonuclease HI has a cavity near Val-74 within the protein core. In order to fill the cavity space, we constructed two mutant proteins, V74L and V74I, in which Val-74 was replaced with either Leu or Ile, respectively. The mutant proteins are stabilized, as revealed by a 2.1-3.7 degrees C increase in the Tm values, as compared to the wild-type protein at pH values of 3.0 and 5.5. The mutant protein V74A, in which Val-74 is replaced with Ala, was also constructed to analyze the reverse effect. The stability of V74A decreases by 7.6 degrees C at pH 3.0 and 12.7 degrees C at pH 5.5 in Tm as compared to those values for the wild-type protein. None of the three mutations significantly affect the enzymatic activity. The crystal structures of V74L and V74I, determined at 1.8-A resolution, are almost identical to that of the wild-type protein, except for the mutation site. In the two mutant proteins, calculation by the Voronoi procedure shows that the cavity volumes around the individual mutation sites are remarkably reduced as compared to that in the wild-type protein. These results indicate that the introduction of a methylene group into the cavity, without causing steric clash, contributes to an increase in the hydrophobic interaction within the protein core and thereby enhances protein stability. We also discuss the role of the Leu side chain, which can assume many different local conformations on a helix without sacrificing thermostability.
About this StructureAbout this Structure
1LAV is a Single protein structure of sequence from Escherichia coli. Active as Ribonuclease H, with EC number 3.1.26.4 Full crystallographic information is available from OCA.
ReferenceReference
Stabilization of Escherichia coli ribonuclease HI by cavity-filling mutations within a hydrophobic core., Ishikawa K, Nakamura H, Morikawa K, Kanaya S, Biochemistry. 1993 Jun 22;32(24):6171-8. PMID:8390295
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