Atomic force microscopy to elicit conformational transitions of ferredoxin-dependent flavin thioredoxin reductases

Marcuello C. ; Frempong G.A. ; Balsera M. ; Medina M. (Universidad de Zaragoza) ; Lostao A.

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.

Idioma: Inglés

DOI: 10.3390/antiox10091437

Año: 2021

Publicado en: Antioxidants 10, 9 (2021), 1437 [19 pp.]
ISSN: 2076-3921

Factor impacto JCR: 7.675 (2021)

Categ. JCR: BIOCHEMISTRY & MOLECULAR BIOLOGY rank: 50 / 297 = 0.168 (2021) - Q1 - T1
Categ. JCR: FOOD SCIENCE & TECHNOLOGY rank: 12 / 144 = 0.083 (2021) - Q1 - T1
Categ. JCR: CHEMISTRY, MEDICINAL rank: 4 / 63 = 0.063 (2021) - Q1 - T1

Factor impacto CITESCORE: 6.5 - Biochemistry, Genetics and Molecular Biology (Q2)

Factor impacto SCIMAGO: 1.008 - Biochemistry (Q1) - Physiology (Q1) - Molecular Biology (Q1) - Clinical Biochemistry (Q1)

Financiación: info:eu-repo/grantAgreement/ES/DGA-FEDER/E35-20R
Financiación: info:eu-repo/grantAgreement/ES/MICINN-AEI/PID2019-103901GB-I00
Financiación: info:eu-repo/grantAgreement/ES/MICINN-AEI/PID2019-110900GB-I00

Tipo y forma: Article (Published version)

Área (Departamento): Área Bioquímica y Biolog.Mole. (Dpto. Bioq.Biolog.Mol. Celular)