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Título: | Atomic force microscopy to elicit conformational transitions of ferredoxin-dependent flavin thioredoxin reductases |
Autor: | Marcuello, Carlos CSIC ORCID; Frempong, Gifty Animwaa; Balsera, Mónica CSIC ORCID ; Medina, Milagros CSIC ORCID; Lostao, Anabel CSIC ORCID | Palabras clave: | Thioredoxin reductase Atomic force microscopy Protein interactions Redox-active disulfide Single-molecule methods Homodimers Flavoproteins |
Fecha de publicación: | 2021 | Editor: | Multidisciplinary Digital Publishing Institute | Citación: | Antioxidants 10(9): 1437 (2021) | Resumen: | 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 | Descripción: | 19 páginas, 8 figuras, 3 tablas complementarias, 4 figuras complementarias | Versión del editor: | http://dx.doi.org/10.3390/antiox10091437 | URI: | http://hdl.handle.net/10261/250810 | DOI: | 10.3390/antiox10091437 | E-ISSN: | 2076-3921 |
Aparece en las colecciones: | (IRNASA) Artículos (INMA) Artículos |
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atomic force microscopy to elicit conformational transitions of ferrodoxin-dependent flavin thioredoxin reductases.pdf | Artículo principal | 8,74 MB | Adobe PDF | Visualizar/Abrir |
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