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

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.

Nanomechanical Study of Enzyme: Coenzyme Complexes: Bipartite Sites in Plastidic Ferredoxin-NADP+ Reductase for the Interaction with NADP+

“…AFM has been previously used to determine the conformational changes in novel flavoenzyme families upon redox partner binding [31]. Nevertheless, the changes in AnFNR upon NADP + binding are not large enough to be visualized and discerned by AFM imaging.…”

Protein Abundance of Drug Transporters in Human Hepatitis C Livers

“…It would also be interesting to combine the observed changes in membrane transporter levels with protein species involved with HCV infection in order to better define the observed interactions. In this framework, fluorescence assays [ 29 , 30 ] to directly monitor the evolution of HCV disease at the cellular level, atomic force microscopy to enable the acquisition of morphological information at the single molecule level [ 31 ], which can be extendable to HCV proteases [ 32 ], docking computational approaches to find flavivirus protease inhibitors [ 33 , 34 ], or integrative methodologies combining X-ray crystallography and cross-linking mass spectrometry studies [ 35 ] are some suitable examples to address this gap in information.…”

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“…Soft dip-pen nanolithography (DPN) is an atomic force microscopy (AFM) setup optimized to precisely control the deposition of biomolecules [31]. AFM is a powerful tool to address biomolecular morphology [32] and the individual biorecognition [33] based on adhesion force. Nevertheless, a self-assembled inkjet printer can be a cost-effective solution for fast biomolecular detection.…”