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A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates
Published on: August 21, 2018
Mutagenesis studies toward understanding allostery in thrombin
Shabir H Qureshi1, Likui Yang, Chandrashekhara Manithody
1Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri 63104, USA.
Biochemistry
|July 31, 2009
Summary
Thrombin
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Thrombin activity is modulated by cofactor binding to exosite-1 (thrombomodulin) and the 225-loop (Na+).
- The energetic linkage between these binding sites and the active site is not fully understood.
Purpose of the Study:
- To investigate the allosteric communication between thrombin's exosite-1, Na+-binding site, and active site.
- To determine if stabilizing these loops affects cofactor binding and catalytic function.
Main Methods:
- Rational design and engineering of two thrombin mutants with disulfide bonds stabilizing exosite-1 (Cys-67/Cys-82) or the Na+-binding site (Cys-217/Cys-224).
- Expression, purification, and characterization of mutants for binding kinetics (thrombomodulin, Na+, heparin) and amidolytic activity.
- Functional assays including fibrinogen clotting and protein C activation.
Main Results:
- The Cys-67/Cys-82 mutant showed no TM binding but normal amidolytic activity.
- The Cys-217/Cys-224 mutant had impaired activity, reduced TM binding affinity, and altered Na+ interaction.
- Neither mutant effectively clotted fibrinogen or activated protein C with TM, but heparin binding was normal.
Conclusions:
- Exosite-2 of thrombin functions independently, but Na+-binding and exosite-1 are energetically linked.
- Na+ allosterically modulates thrombin's catalytic residue, while TM does not directly affect its conformation.
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