Conformational flexibility of a model protein upon immobilization on self-assembled monolayers.
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.
Bigdeli
Saharnaz
S
Stanford Genome Technology Center, 855 California Ave, Palo Alto, California 94304, USA.
Talasaz
AmirAli H
AH
Ståhl
Patrik
P
Persson
Henrik H J
HH
Ronaghi
Mostafa
M
Davis
Ronald W
RW
Nemat-Gorgani
Mohsen
M
D000327:Adsorption; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D020099:Coated Materials, Biocompatible / Q000737:chemistry*; D003198:Computer Simulation; D004789:Enzyme Activation; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry; D005969:Glutamate Dehydrogenase / Q000737:chemistry* / Q000648:ultrastructure*; D006046:Gold / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation
D020099:Coated Materials, Biocompatible; D004800:Enzymes, Immobilized; D006046:Gold; D005969:Glutamate Dehydrogenase