Conformational flexibility of a model protein upon immobilization on self-assembled monolayers
Autor: | Mostafa Ronaghi, Ronald W. Davis, AmirAli Talasaz, Saharnaz Bigdeli, Patrik Ståhl, Henrik H. J. Persson, Mohsen Nemat-Gorgani |
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Rok vydání: | 2008 |
Předmět: |
Models
Molecular Protein Conformation Stereochemistry Allosteric regulation Bioengineering Applied Microbiology and Biotechnology Protein structure Coated Materials Biocompatible Glutamate Dehydrogenase Enzyme Stability Animals Computer Simulation chemistry.chemical_classification Binding Sites biology Chemistry Glutamate dehydrogenase Chemical modification Enzymes Immobilized Enzyme Activation Enzyme Models Chemical Allosteric enzyme Covalent bond biology.protein Biophysics Cattle Chemical binding Adsorption Gold Protein Binding Biotechnology |
Zdroj: | Biotechnology and Bioengineering. 100:19-27 |
ISSN: | 1097-0290 0006-3592 |
Popis: | The present study reports on the retention of conformational flexibility of a model allosteric protein upon immobilization on self-assembled monolayers (SAMs) on gold. Organothiolated SAMs of different compositions were utilized for adsorptive and covalent attachment of bovine liver glutamate dehydrogenase (GDH), a well-characterized allosteric enzyme. Sensitive fluorimetric assays were developed to determine immobilization capacity, specific activity, and allosteric properties of the immobilized preparations as well as the potential for repeated use and continuous catalytic transformations. The allosteric response of the free and immobilized forms towards ADP, L-leucine and high concentrations of NAD+, some of the well-known activators for this enzyme, were determined and compared. The enzyme immobilized by adsorption or chemical binding responded similarly to the activators with a greater degree of activation, as compared to the free form. Also loss of activity involving the two immobilization procedures were similar, suggesting that residues essential for catalytic activity or allosteric properties of GDH remained unchanged in the course of chemical modification. A recently established method was used to predict GDH orientation upon immobilization, which was found to explain some of the experimental results presented. The general significance of these observations in connection with retention of native properties of protein structures upon immobilization on SAMs is discussed. Biotechnol. Bioeng. 2008;100: 19–27. © 2007 Wiley Periodicals, Inc. |
Databáze: | OpenAIRE |
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