70295 20230313141235.0 doi 10.1093/protein/gzs086 sideral 80146 ART-2012-80146 eng Marcuello, C. An efficient method for enzyme immobilization evidenced by atomic force microscopy 2012 Access copy available to the general public Unrestricted 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-NADP+ 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. info:eu-repo/grantAgreement/ES/DGA/B18 info:eu-repo/grantAgreement/ES/MEC/BIO2006-09178-C02 info:eu-repo/grantAgreement/ES/MICINN/BIO2010-14983 info:eu-repo/semantics/openAccess All rights reserved http://www.europeana.eu/rights/rr-f/ 2.588 2012 BIOTECHNOLOGY & APPLIED MICROBIOLOGY 58 / 159 = 0.365 2012 Q2 T2 BIOCHEMISTRY & MOLECULAR BIOLOGY 164 / 290 = 0.566 2012 Q3 T2 info:eu-repo/semantics/article info:eu-repo/semantics/acceptedVersion De Miguel, R. Gómez-Moreno, C. Martínez-Júlvez, M. Lostao, A. Universidad de Zaragoza (orcid)0000-0001-7460-5916 1002 060 Universidad de Zaragoza Dpto. Bioq.Biolog.Mol. Celular Área Bioquímica y Biolog.Mole. 25, 11 (2012), 715-723 PROTEIN ENG DES SEL PROTEIN ENGINEERING DESIGN & SELECTION 1741-0126 406033 http://zaguan.unizar.es/record/70295/files/texto_completo.pdf Postprint 40186 http://zaguan.unizar.es/record/70295/files/texto_completo.jpg?subformat=icon icon Postprint oai:zaguan.unizar.es:70295 articulos driver 2023-03-13-13:54:37 ARTICLE