Skip to main content

Binding and signaling of surface-immobilized reagentless fluorescent biosensors derived from periplasmic binding proteins.

Publication ,  Journal Article
de Lorimier, RM; Tian, Y; Hellinga, HW
Published in: Protein Sci
August 2006

Development of biosensor devices typically requires incorporation of the molecular recognition element into a solid surface for interfacing with a signal detector. One approach is to immobilize the signal transducing protein directly on a solid surface. Here we compare the effects of two direct immobilization methods on ligand binding, kinetics, and signal transduction of reagentless fluorescent biosensors based on engineered periplasmic binding proteins. We used thermostable ribose and glucose binding proteins cloned from Thermoanaerobacter tengcongensis and Thermotoga maritima, respectively. To test the behavior of these proteins in semispecifically oriented layers, we covalently modified lysine residues with biotin or sulfhydryl functions, and attached the conjugates to plastic surfaces derivatized with streptavidin or maleimide, respectively. The immobilized proteins retained ligand binding and signal transduction but with adversely affected affinities and signal amplitudes for the thiolated, but not the biotinylated, proteins. We also immobilized these proteins in a more specifically oriented layer to maleimide-derivatized plates using a His(2)Cys(2) zinc finger domain fused at either their N or C termini. Proteins immobilized this way either retained, or displayed enhanced, ligand affinity and signal amplitude. In all cases tested ligand binding by immobilized proteins is reversible, as demonstrated by several iterations of ligand loading and elution. The kinetics of ligand exchange with the immobilized proteins are on the order of seconds.

Duke Scholars

Altmetric Attention Stats
Dimensions Citation Stats

Published In

Protein Sci

DOI

ISSN

0961-8368

Publication Date

August 2006

Volume

15

Issue

8

Start / End Page

1936 / 1944

Location

United States

Related Subject Headings

  • Zinc Fingers
  • Thermotoga maritima
  • Thermoanaerobacter
  • Surface Properties
  • Sulfhydryl Compounds
  • Streptavidin
  • Signal Transduction
  • Ribose
  • Periplasmic Binding Proteins
  • Monosaccharide Transport Proteins
 

Citation

APA
Chicago
ICMJE
MLA
NLM
de Lorimier, R. M., Tian, Y., & Hellinga, H. W. (2006). Binding and signaling of surface-immobilized reagentless fluorescent biosensors derived from periplasmic binding proteins. Protein Sci, 15(8), 1936–1944. https://doi.org/10.1110/ps.062261606
Lorimier, Robert M. de, Yaji Tian, and Homme W. Hellinga. “Binding and signaling of surface-immobilized reagentless fluorescent biosensors derived from periplasmic binding proteins.Protein Sci 15, no. 8 (August 2006): 1936–44. https://doi.org/10.1110/ps.062261606.
de Lorimier, Robert M., et al. “Binding and signaling of surface-immobilized reagentless fluorescent biosensors derived from periplasmic binding proteins.Protein Sci, vol. 15, no. 8, Aug. 2006, pp. 1936–44. Pubmed, doi:10.1110/ps.062261606.

Published In

Protein Sci

DOI

ISSN

0961-8368

Publication Date

August 2006

Volume

15

Issue

8

Start / End Page

1936 / 1944

Location

United States

Related Subject Headings

  • Zinc Fingers
  • Thermotoga maritima
  • Thermoanaerobacter
  • Surface Properties
  • Sulfhydryl Compounds
  • Streptavidin
  • Signal Transduction
  • Ribose
  • Periplasmic Binding Proteins
  • Monosaccharide Transport Proteins