1rxh: Difference between revisions
New page: left|200px<br /><applet load="1rxh" size="450" color="white" frame="true" align="right" spinBox="true" caption="1rxh, resolution 2.9Å" /> '''Crystal structure of ... |
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[[Image:1rxh.gif|left|200px]]<br /><applet load="1rxh" size=" | [[Image:1rxh.gif|left|200px]]<br /><applet load="1rxh" size="350" color="white" frame="true" align="right" spinBox="true" | ||
caption="1rxh, resolution 2.9Å" /> | caption="1rxh, resolution 2.9Å" /> | ||
'''Crystal structure of streptavidin mutant L124R (M1) complexed with biotinyl p-nitroanilide (BNI)'''<br /> | '''Crystal structure of streptavidin mutant L124R (M1) complexed with biotinyl p-nitroanilide (BNI)'''<br /> | ||
==Overview== | ==Overview== | ||
Avidin enhances the hydrolysis of biotinyl p-nitrophenyl ester (BNP) under | Avidin enhances the hydrolysis of biotinyl p-nitrophenyl ester (BNP) under mild alkaline conditions, whereas streptavidin prevents hydrolysis of BNP up to pH 12. Recently, we imposed hydrolytic activity on streptavidin by rational mutagenesis, based on the molecular elements responsible for the hydrolysis by avidin. Three mutants were designed, whereby the desired features, the distinctive L124R point mutation (M1), the L3,4 loop replacement (M2), and the combined mutation (M3), were transferred from avidin to streptavidin. The crystal structures of the mutants, in complex with biotinyl p-nitroanilide (BNA), the stable amide analogue of BNP, were determined. The results demonstrate that the point mutation alone has little effect on hydrolysis, and BNA exhibits a conformation similar to that of streptavidin. Substitution of a lengthier L3,4 loop (from avidin to streptavidin), resulted in an open conformation, thus exposing the ligand to solvent. Moreover, the amide bond of BNA was flipped relative to that of the streptavidin and M1 complexes, thus deflecting the nitro group toward Lys-121. Consequently, the leaving group potential of the nitrophenyl group of BNP is increased, and M2 hydrolyzes BNP at pH values >8.5. To better emulate the hydrolytic potential of avidin, M3 was required. The combination of loop replacement and point mutation served to further increase the leaving group potential by interaction of the nitro group with Arg-124 and Lys-121. The information derived from this study may provide insight into the design of enzymes and transfer of desired properties among homologous proteins. | ||
==About this Structure== | ==About this Structure== | ||
1RXH is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Streptomyces_avidinii Streptomyces avidinii] with BNI as [http://en.wikipedia.org/wiki/ligand ligand]. Full crystallographic information is available from [http:// | 1RXH is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Streptomyces_avidinii Streptomyces avidinii] with <scene name='pdbligand=BNI:'>BNI</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1RXH OCA]. | ||
==Reference== | ==Reference== | ||
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[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Streptomyces avidinii]] | [[Category: Streptomyces avidinii]] | ||
[[Category: Bayer, E | [[Category: Bayer, E A.]] | ||
[[Category: Eisenberg-Domovich, Y.]] | [[Category: Eisenberg-Domovich, Y.]] | ||
[[Category: Livnah, O.]] | [[Category: Livnah, O.]] | ||
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[[Category: streptavidin]] | [[Category: streptavidin]] | ||
''Page seeded by [http:// | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 14:55:36 2008'' | ||
Revision as of 15:55, 21 February 2008
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Crystal structure of streptavidin mutant L124R (M1) complexed with biotinyl p-nitroanilide (BNI)
OverviewOverview
Avidin enhances the hydrolysis of biotinyl p-nitrophenyl ester (BNP) under mild alkaline conditions, whereas streptavidin prevents hydrolysis of BNP up to pH 12. Recently, we imposed hydrolytic activity on streptavidin by rational mutagenesis, based on the molecular elements responsible for the hydrolysis by avidin. Three mutants were designed, whereby the desired features, the distinctive L124R point mutation (M1), the L3,4 loop replacement (M2), and the combined mutation (M3), were transferred from avidin to streptavidin. The crystal structures of the mutants, in complex with biotinyl p-nitroanilide (BNA), the stable amide analogue of BNP, were determined. The results demonstrate that the point mutation alone has little effect on hydrolysis, and BNA exhibits a conformation similar to that of streptavidin. Substitution of a lengthier L3,4 loop (from avidin to streptavidin), resulted in an open conformation, thus exposing the ligand to solvent. Moreover, the amide bond of BNA was flipped relative to that of the streptavidin and M1 complexes, thus deflecting the nitro group toward Lys-121. Consequently, the leaving group potential of the nitrophenyl group of BNP is increased, and M2 hydrolyzes BNP at pH values >8.5. To better emulate the hydrolytic potential of avidin, M3 was required. The combination of loop replacement and point mutation served to further increase the leaving group potential by interaction of the nitro group with Arg-124 and Lys-121. The information derived from this study may provide insight into the design of enzymes and transfer of desired properties among homologous proteins.
About this StructureAbout this Structure
1RXH is a Single protein structure of sequence from Streptomyces avidinii with as ligand. Full crystallographic information is available from OCA.
ReferenceReference
Structural elements responsible for conversion of streptavidin to a pseudoenzyme., Eisenberg-Domovich Y, Pazy Y, Nir O, Raboy B, Bayer EA, Wilchek M, Livnah O, Proc Natl Acad Sci U S A. 2004 Apr 20;101(16):5916-21. Epub 2004 Apr 12. PMID:15079055
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