000070295 001__ 70295
000070295 005__ 20180509095017.0
000070295 0247_ $$2doi$$a10.1093/protein/gzs086
000070295 0248_ $$2sideral$$a80146
000070295 037__ $$aART-2012-80146
000070295 041__ $$aeng
000070295 100__ $$aMarcuello, C.
000070295 245__ $$aAn efficient method for enzyme immobilization evidenced by atomic force microscopy
000070295 260__ $$c2012
000070295 5060_ $$aAccess copy available to the general public$$fUnrestricted
000070295 5203_ $$aImmobilization 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.
000070295 536__ $$9info:eu-repo/grantAgreement/ES/DGA/B18$$9info:eu-repo/grantAgreement/ES/MEC/BIO2006-09178-C02$$9info:eu-repo/grantAgreement/ES/MICINN/BIO2010-14983
000070295 540__ $$9info:eu-repo/semantics/openAccess$$aAll rights reserved$$uhttp://www.europeana.eu/rights/rr-f/
000070295 590__ $$a2.588$$b2012
000070295 591__ $$aBIOTECHNOLOGY & APPLIED MICROBIOLOGY$$b58 / 156 = 0.372$$c2012$$dQ2$$eT2
000070295 591__ $$aBIOCHEMISTRY & MOLECULAR BIOLOGY$$b164 / 288 = 0.569$$c2012$$dQ3$$eT2
000070295 655_4 $$ainfo:eu-repo/semantics/article$$vinfo:eu-repo/semantics/acceptedVersion
000070295 700__ $$aDe Miguel, R.
000070295 700__ $$aGómez-Moreno, C.
000070295 700__ $$aMartínez-Júlvez, M.
000070295 700__ $$0(orcid)0000-0001-7460-5916$$aLostao, A.$$uUniversidad de Zaragoza
000070295 7102_ $$11002$$2060$$aUniversidad de Zaragoza$$bDepartamento de Bioquímica y Biología Molecular y Celular$$cBioquímica y Biología Molecular
000070295 773__ $$g25, 11 (2012), 715-723$$pPROTEIN ENG DES SEL$$tPROTEIN ENGINEERING DESIGN & SELECTION$$x1741-0126
000070295 8564_ $$s406033$$uhttps://zaguan.unizar.es/record/70295/files/texto_completo.pdf$$yPostprint
000070295 8564_ $$s40186$$uhttps://zaguan.unizar.es/record/70295/files/texto_completo.jpg?subformat=icon$$xicon$$yPostprint
000070295 909CO $$ooai:zaguan.unizar.es:70295$$particulos$$pdriver
000070295 951__ $$a2018-05-09-09:27:00
000070295 980__ $$aARTICLE