3aky
From Proteopedia
(New page: 200px<br /><applet load="3aky" size="450" color="white" frame="true" align="right" spinBox="true" caption="3aky, resolution 2.23Å" /> '''STABILITY, ACTIVITY ...) |
|||
| Line 1: | Line 1: | ||
| - | [[Image:3aky.jpg|left|200px]]<br /><applet load="3aky" size=" | + | [[Image:3aky.jpg|left|200px]]<br /><applet load="3aky" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="3aky, resolution 2.23Å" /> | caption="3aky, resolution 2.23Å" /> | ||
'''STABILITY, ACTIVITY AND STRUCTURE OF ADENYLATE KINASE MUTANTS'''<br /> | '''STABILITY, ACTIVITY AND STRUCTURE OF ADENYLATE KINASE MUTANTS'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Sequence/structure relationships have been explored by site-directed | + | Sequence/structure relationships have been explored by site-directed mutagenesis using a structurally known adenylate kinase. In particular the effects of helix capping and nonpolar core expansion on thermodynamic stability have been analyzed. Six point mutations were produced and characterized by SDS/PAGE, native PAGE, isoelectric focussing, electrophoretic titration, enzyme kinetics, and X-ray structure analysis. Heat-denaturation experiments yielded melting temperatures Tm and melting enthalpy changes delta Hm. The heat capacity change delta Cp of the wild-type enzyme was determined by guanidine hydrochloride denaturation in conjunction with Tm and delta Hm. Using the wild-type delta Cp value, Gibbs free energy changes delta G at room temperature were calculated for all mutants. Four mutants were designed for helix capping stabilization, but only one of them showed such an effect. Because of electrostatic interference with the induced-fit motion, one mutant decreased the catalytic activity strongly. Two mutants expanded nonpolar cores causing destabilization. The mutant with the lower stability could be crystallized and subjected to an X-ray analysis at 223-pm resolution which showed the structural changes. The enzyme was stabilized by adding a -Pro-His-His tail to the C-terminal alpha-helix for nickel-chelate chromatography. This addition constitutes a helix cap. Taken together, the results demonstrate that stabilization by helix capping is difficult to achieve because the small positive effect is drowned by adverse mutational disruption. Further addition of atoms to nonpolar cores destabilized the protein, although the involved geometry changes were very small, demonstrating the importance of efficient packing. |
==About this Structure== | ==About this Structure== | ||
| - | 3AKY is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Saccharomyces_cerevisiae Saccharomyces cerevisiae] with AP5 and IMD as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Adenylate_kinase Adenylate kinase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.7.4.3 2.7.4.3] Full crystallographic information is available from [http:// | + | 3AKY is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Saccharomyces_cerevisiae Saccharomyces cerevisiae] with <scene name='pdbligand=AP5:'>AP5</scene> and <scene name='pdbligand=IMD:'>IMD</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Adenylate_kinase Adenylate kinase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.7.4.3 2.7.4.3] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3AKY OCA]. |
==Reference== | ==Reference== | ||
| Line 15: | Line 15: | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Abele, U.]] | [[Category: Abele, U.]] | ||
| - | [[Category: Schulz, G | + | [[Category: Schulz, G E.]] |
[[Category: AP5]] | [[Category: AP5]] | ||
[[Category: IMD]] | [[Category: IMD]] | ||
| Line 21: | Line 21: | ||
[[Category: myokinase]] | [[Category: myokinase]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 19:02:41 2008'' |
Revision as of 17:02, 21 February 2008
|
STABILITY, ACTIVITY AND STRUCTURE OF ADENYLATE KINASE MUTANTS
Overview
Sequence/structure relationships have been explored by site-directed mutagenesis using a structurally known adenylate kinase. In particular the effects of helix capping and nonpolar core expansion on thermodynamic stability have been analyzed. Six point mutations were produced and characterized by SDS/PAGE, native PAGE, isoelectric focussing, electrophoretic titration, enzyme kinetics, and X-ray structure analysis. Heat-denaturation experiments yielded melting temperatures Tm and melting enthalpy changes delta Hm. The heat capacity change delta Cp of the wild-type enzyme was determined by guanidine hydrochloride denaturation in conjunction with Tm and delta Hm. Using the wild-type delta Cp value, Gibbs free energy changes delta G at room temperature were calculated for all mutants. Four mutants were designed for helix capping stabilization, but only one of them showed such an effect. Because of electrostatic interference with the induced-fit motion, one mutant decreased the catalytic activity strongly. Two mutants expanded nonpolar cores causing destabilization. The mutant with the lower stability could be crystallized and subjected to an X-ray analysis at 223-pm resolution which showed the structural changes. The enzyme was stabilized by adding a -Pro-His-His tail to the C-terminal alpha-helix for nickel-chelate chromatography. This addition constitutes a helix cap. Taken together, the results demonstrate that stabilization by helix capping is difficult to achieve because the small positive effect is drowned by adverse mutational disruption. Further addition of atoms to nonpolar cores destabilized the protein, although the involved geometry changes were very small, demonstrating the importance of efficient packing.
About this Structure
3AKY is a Single protein structure of sequence from Saccharomyces cerevisiae with and as ligands. Active as Adenylate kinase, with EC number 2.7.4.3 Full crystallographic information is available from OCA.
Reference
Stability, activity and structure of adenylate kinase mutants., Spuergin P, Abele U, Schulz GE, Eur J Biochem. 1995 Jul 15;231(2):405-13. PMID:7635152
Page seeded by OCA on Thu Feb 21 19:02:41 2008
