Atomic Force Microscopy to Elicit Conformational Transitions of Ferredoxin-Dependent Flavin Thioredoxin Reductases.

Carlos Marcuello,Milagros Medina,Mónica Balsera,Gifty Animwaa Frempong,Anabel Lostao

doi:10.3390/antiox10091437

Abstract

Flavin and redox-active disulfide domains of ferredoxin-dependent flavin thioredoxin reductase (FFTR) homodimers should pivot between flavin-oxidizing (FO) and flavin-reducing (FR) conformations during catalysis, but only FR conformations have been detected by X-ray diffraction and scattering techniques. Atomic force microscopy (AFM) is a single-molecule technique that allows the observation of individual biomolecules with sub-nm resolution in near-native conditions in real-time, providing sampling of molecular properties distributions and identification of existing subpopulations. Here, we show that AFM is suitable to evaluate FR and FO conformations. In agreement with imaging under oxidizing condition, only FR conformations are observed for Gloeobacter violaceus FFTR (GvFFTR) and isoform 2 of Clostridium acetobutylicum FFTR (CaFFTR2). Nonetheless, different relative dispositions of the redox-active disulfide and FAD-binding domains are detected for FR homodimers, indicating a dynamic disposition of disulfide domains regarding the central protein core in solution. This study also shows that AFM can detect morphological changes upon the interaction of FFTRs with their protein partners. In conclusion, this study paves way for using AFM to provide complementary insight into the FFTR catalytic cycle at pseudo-physiological conditions. However, future approaches for imaging of FO conformations will require technical developments with the capability of maintaining the FAD-reduced state within the protein during AFM scanning.

Highlights

The thioredoxin system is responsible for the reduction of disulfide bonds in target proteins under physiological conditions
C-terminal tail has been deleted from Gloeobacter violaceus FFTR (GvFFTR), Table A1) samples on mica surfaces by Atomic force microscopy (AFM), we found that for all of them quantification of relative volumes was useful to identify subpopulations (Figures 2 and A2, and Table A2)
GvFFTR, CaFFTR2, and GvDDOR samples were imaged after incubation with a detergent solution formed by SDS and Tween 20 in a concentration low enough to disrupt protein–protein interactions but not to denature proteins [15]

Summary

Introduction

The thioredoxin system is responsible for the reduction of disulfide bonds in target proteins under physiological conditions. It is widely distributed in most types of cells, constituting one of the central antioxidants systems [1]. It is composed of a reduced substrate, a thioredoxin reductase (TR), and a thioredoxin (Trx), a conserved protein that contains an invariant WCGPC motif with the two Cys forming a redox-active intramolecular disulfide [2]. TR catalyzes the reduction of the disulfide in Trx using electrons derived from the reduced substrate, typically in the form of NAD(P)H or ferredoxin (Fdx) [3]. Among the different types of TRs [1], Fdx-dependent flavin

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Discussion

Conclusion