Article

Conformational flexibility of a model protein upon immobilization on self-assembled monolayers†

  1. Saharnaz Bigdeli1,
  2. AmirAli H. Talasaz1,2,
  3. Patrik Ståhl1,
  4. Henrik H.J. Persson1,
  5. Mostafa Ronaghi1,
  6. Ronald W. Davis1,
  7. Mohsen Nemat-Gorgani1,*

Article first published online: 13 DEC 2007

DOI: 10.1002/bit.21724

Issue

Biotechnology and Bioengineering

Biotechnology and Bioengineering

Volume 100, Issue 1, pages 19–27, 1 May 2008

Additional Information

How to Cite

Bigdeli, S., Talasaz, A. H., Ståhl, P., Persson, H. H.J., Ronaghi, M., Davis, R. W. and Nemat-Gorgani, M. (2008), Conformational flexibility of a model protein upon immobilization on self-assembled monolayers. Biotechnol. Bioeng., 100: 19–27. doi: 10.1002/bit.21724

Author Information

  1. 1

    Stanford Genome Technology Center, 855 California Ave, Palo Alto, California 94304; telephone: 650-812-1961; fax: 650-812-1975

  2. 2

    Electrical Engineering Department, Stanford University, California 94305

*Stanford Genome Technology Center, 855 California Ave, Palo Alto, California 94304; telephone: 650-812-1961; fax: 650-812-1975.

  1. †

    Saharnaz Bigdeli and AmirAli H. Talasaz contributed equally to this work.

Publication History

  1. Issue published online: 25 MAR 2008
  2. Article first published online: 13 DEC 2007
  3. Manuscript Accepted: 29 OCT 2007
  4. Manuscript Revised: 14 SEP 2007
  5. Manuscript Received: 18 JUN 2007

Funded by

  • National Institutes of Health. Grant Number: PO1 HG000205

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Keywords:

  • glutamate dehydrogenase;
  • allosteric effectors;
  • self-assembled monolayer;
  • conformational flexibility;
  • immobilization;
  • orientation

Abstract

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.

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