1n3w
From Proteopedia
(New page: 200px<br /><applet load="1n3w" size="450" color="white" frame="true" align="right" spinBox="true" caption="1n3w, resolution 2.60Å" /> '''Engineered High-Affi...) |
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| - | [[Image:1n3w.jpg|left|200px]]<br /><applet load="1n3w" size=" | + | [[Image:1n3w.jpg|left|200px]]<br /><applet load="1n3w" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1n3w, resolution 2.60Å" /> | caption="1n3w, resolution 2.60Å" /> | ||
'''Engineered High-Affinity Maltose-Binding Protein'''<br /> | '''Engineered High-Affinity Maltose-Binding Protein'''<br /> | ||
==Overview== | ==Overview== | ||
| - | The affinity of maltose-binding protein (MBP) for maltose and related | + | The affinity of maltose-binding protein (MBP) for maltose and related carbohydrates was greatly increased by removal of groups in the interface opposite the ligand binding cleft. The wild-type protein has a KD of 1200 nM for maltose; mutation of residues Met-321 and Gln-325, both to alanine, resulted in a KD for maltose of 70 nM; deletion of 4 residues, Glu-172, Asn-173, Lys-175, and Tyr-176, which are part of a poorly ordered loop, results in a KD for maltose of 110 nM. Combining the mutations yields an increased affinity for maltodextrins and a KD of 6 nM for maltotriose. Comparison of ligand binding by the mutants, using surface plasmon resonance spectroscopy, indicates that decreases in the off-rate are responsible for the increased affinity. Small-angle x-ray scattering was used to demonstrate that the mutations do not significantly affect the solution conformation of MBP in either the presence or absence of maltose. The crystal structures of selected mutants showed that the mutations do not cause significant structural changes in either the closed or open conformation of MBP. These studies show that interactions in the interface opposite the ligand binding cleft, which we term the "balancing interface," are responsible for modulating the affinity of MBP for its ligand. Our results are consistent with a model in which the ligand-bound protein alternates between the closed and open conformations, and removal of interactions in the balancing interface decreases the stability of the open conformation, without affecting the closed conformation. |
==About this Structure== | ==About this Structure== | ||
| - | 1N3W is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with MAL as [http://en.wikipedia.org/wiki/ligand ligand]. Full crystallographic information is available from [http:// | + | 1N3W is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with <scene name='pdbligand=MAL:'>MAL</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1N3W OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Escherichia coli]] | [[Category: Escherichia coli]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Shilton, B | + | [[Category: Shilton, B H.]] |
| - | [[Category: Telmer, P | + | [[Category: Telmer, P G.]] |
[[Category: MAL]] | [[Category: MAL]] | ||
[[Category: engineered]] | [[Category: engineered]] | ||
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[[Category: mbpdel-liganded]] | [[Category: mbpdel-liganded]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 14:01:58 2008'' |
Revision as of 12:01, 21 February 2008
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Engineered High-Affinity Maltose-Binding Protein
Overview
The affinity of maltose-binding protein (MBP) for maltose and related carbohydrates was greatly increased by removal of groups in the interface opposite the ligand binding cleft. The wild-type protein has a KD of 1200 nM for maltose; mutation of residues Met-321 and Gln-325, both to alanine, resulted in a KD for maltose of 70 nM; deletion of 4 residues, Glu-172, Asn-173, Lys-175, and Tyr-176, which are part of a poorly ordered loop, results in a KD for maltose of 110 nM. Combining the mutations yields an increased affinity for maltodextrins and a KD of 6 nM for maltotriose. Comparison of ligand binding by the mutants, using surface plasmon resonance spectroscopy, indicates that decreases in the off-rate are responsible for the increased affinity. Small-angle x-ray scattering was used to demonstrate that the mutations do not significantly affect the solution conformation of MBP in either the presence or absence of maltose. The crystal structures of selected mutants showed that the mutations do not cause significant structural changes in either the closed or open conformation of MBP. These studies show that interactions in the interface opposite the ligand binding cleft, which we term the "balancing interface," are responsible for modulating the affinity of MBP for its ligand. Our results are consistent with a model in which the ligand-bound protein alternates between the closed and open conformations, and removal of interactions in the balancing interface decreases the stability of the open conformation, without affecting the closed conformation.
About this Structure
1N3W is a Single protein structure of sequence from Escherichia coli with as ligand. Full crystallographic information is available from OCA.
Reference
Insights into the conformational equilibria of maltose-binding protein by analysis of high affinity mutants., Telmer PG, Shilton BH, J Biol Chem. 2003 Sep 5;278(36):34555-67. Epub 2003 Jun 6. PMID:12794084
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