Atomic Force Microscopy applied to the Study of single Flavoprotein Complexes
A. Lostao, J.Sotres, R. de Miguel, M. Martínez-Júlvez, C. Gómez-Moreno and A.M. Baró. Atomic Force Microscopy applied to the Study of single Flavoprotein Complexes. Flavins and Flavoproteins. Zaragoza: Prensas Universitarias, 2008. Vol.16, p.589-594
Atomic force microscopy (AFM) is a technique capable to study biological recognition processes at the single-molecule level. AFM is able to measure forces down to the piconewton range in a liquid environment. In the last years, interaction forces of different protein-ligand complexes have been measured by force spectroscopy (FS). The methodology measures the so-called force scans, in which a ligand molecule is bound to the tip, at the end of the cantilever, and the sample contains the receptor molecule. Typically, the immobilization of both molecules is carried out in a non-oriented manner being the protein at the substrate bound as a monolayer. Best results have occurred when the ligand is connected to the tip through a flexible polymer of sufficient length and motility [1].
In this work we introduce some important methodology advances to measure single flavoprotein complexes, being a promising field to study these proteins, where only a few attempts have been done [2]. We describe the AFM-FS conditions to measure interaction forces on a single-complex, studied with the model system avidin-biotin that will then apply to different flavoprotein complexes. We have developed new strategies of oriented immobilization of two redox partners, the flavin-dependent Ferredoxin-NADP+ reductase (FNR) and the Ferredoxin (Fd). The FNR catalyses the transfer of two electrons from two independent Fd molecules, previously reduced by Photosystem I (PSI), to NADP+. This reaction requires formation of a complex that allows the optimal orientation between the redox centres of both, FNR and Fd, for the subsequent electron transfer (ET) [3].
Atomic force microscopy (AFM) is a technique capable to study biological recognition processes at the single-molecule level. AFM is able to measure forces down to the piconewton range in a liquid environment. In the last years, interaction forces of different protein-ligand complexes have been measured by force spectroscopy (FS). The methodology measures the so-called force scans, in which a ligand molecule is bound to the tip, at the end of the cantilever, and the sample contains the receptor molecule. Typically, the immobilization of both molecules is carried out in a non-oriented manner being the protein at the substrate bound as a monolayer. Best results have occurred when the ligand is connected to the tip through a flexible polymer of sufficient length and motility [1].
In this work we introduce some important methodology advances to measure single flavoprotein complexes, being a promising field to study these proteins, where only a few attempts have been done [2]. We describe the AFM-FS conditions to measure interaction forces on a single-complex, studied with the model system avidin-biotin that will then apply to different flavoprotein complexes. We have developed new strategies of oriented immobilization of two redox partners, the flavin-dependent Ferredoxin-NADP+ reductase (FNR) and the Ferredoxin (Fd). The FNR catalyses the transfer of two electrons from two independent Fd molecules, previously reduced by Photosystem I (PSI), to NADP+. This reaction requires formation of a complex that allows the optimal orientation between the redox centres of both, FNR and Fd, for the subsequent electron transfer (ET) [3].