An efficient method for enzyme immobilization evidenced by atomic force microscopy

C. Marcuello, R. de Miguel, M. Martínez-Júlvez C. Gómez-Moreno, A. Lostao. An efficient method for enzyme immobilization evidenced by atomic force microscopy. Protein Engineering, Design and Selection. 2012, Vol. 25 (11), p. 715-2012.

Immobilization of proteins in a functionally active form
and proper orientation is fundamental for effective
surface-based protein analysis. A new method is presented
for the controlled and oriented immobilization of
ordered monolayers of enzymes whose interaction site
had been protected using the protein ligand. The utility
of this method was demonstrated by analyzing the interactions
between the enzyme ferredoxin-NADP1 reductase
(FNR) and its redox partner ferredoxin (Fd). The quality
of the procedure was deeply evaluated through enzymatic
assays and atomic force microscopy. Single-molecule
force spectroscopy revealed that site-specifically targeted
FNR samples increased the ratio of recognition events 4-
fold with regard to the standard randomly modified FNR
samples. The results were corroborated using the cytochrome
c reductase activity that gave an increase on
surface between 6 and 12 times for the site-specifically
targeted FNR samples. The activity in solution for the
enzyme labeled from the complex was similar to that
exhibited by wild-type FNR while FNR randomly tagged
showed a 3-fold decrease. This indicates that random targeting
protocols affect not only the efficiency of immobilized
proteins to recognize their ligands but also their
general functionality. The present methodology is
expected to find wide applications in surface-based
protein–protein interactions biosensors, single-molecule
analysis, bioelectronics or drug screening.