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

Antioxidants ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 1437
Author(s):  
Carlos Marcuello ◽  
Gifty Animwaa Frempong ◽  
Mónica Balsera ◽  
Milagros Medina ◽  
Anabel Lostao
Keyword(s):  
Atomic Force Microscopy ◽  
Single Molecule ◽  
Morphological Changes ◽  
Thioredoxin Reductase ◽  
Force Microscopy ◽  
Binding Domains ◽  
Technical Developments ◽  
Atomic Force ◽  
Redox Active ◽  
Central Protein

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.

Atomic Force Microscopy (AFM) for Topography and Recognition Imaging at Single Molecule Level

2013 ◽  
pp. 102-112
Author(s):  
Memed Duman ◽  
Andreas Ebner ◽  
Christian Rankl ◽  
Jilin Tang ◽  
Lilia A. Chtcheglova ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Single Molecule ◽  
Single Molecule Level ◽  
Force Microscopy ◽  
Atomic Force ◽  
Recognition Imaging

scholarly journals Atomic Force Microscopy Investigation of the Interactions between the MCM Helicase and DNA

Materials ◽  
2021 ◽  
Vol 14 (3) ◽  
pp. 687
Author(s):  
Amna Abdalla Mohammed Khalid ◽  
Pietro Parisse ◽  
Barbara Medagli ◽  
Silvia Onesti ◽  
Loredana Casalis
Keyword(s):  
Atomic Force Microscopy ◽  
Nucleic Acid ◽  
Single Molecule ◽  
Protein Complex ◽  
The Other ◽  
Contour Length ◽  
Force Microscopy ◽  
Atomic Force ◽  
Mcm Complex ◽  
Dna Double Helix

The MCM (minichromosome maintenance) protein complex forms an hexameric ring and has a key role in the replication machinery of Eukaryotes and Archaea, where it functions as the replicative helicase opening up the DNA double helix ahead of the polymerases. Here, we present a study of the interaction between DNA and the archaeal MCM complex from Methanothermobacter thermautotrophicus by means of atomic force microscopy (AFM) single molecule imaging. We first optimized the protocol (surface treatment and buffer conditions) to obtain AFM images of surface-equilibrated DNA molecules before and after the interaction with the protein complex. We discriminated between two modes of interaction, one in which the protein induces a sharp bend in the DNA, and one where there is no bending. We found that the presence of the MCM complex also affects the DNA contour length. A possible interpretation of the observed behavior is that in one case the hexameric ring encircles the dsDNA, while in the other the nucleic acid wraps on the outside of the ring, undergoing a change of direction. We confirmed this topographical assignment by testing two mutants, one affecting the N-terminal β-hairpins projecting towards the central channel, and thus preventing DNA loading, the other lacking an external subdomain and thus preventing wrapping. The statistical analysis of the distribution of the protein complexes between the two modes, together with the dissection of the changes of DNA contour length and binding angle upon interaction, for the wild type and the two mutants, is consistent with the hypothesis. We discuss the results in view of the various modes of nucleic acid interactions that have been proposed for both archaeal and eukaryotic MCM complexes.

Substrate Specificity of Sugar Transport by Rabbit SGLT1:  Single-Molecule Atomic Force Microscopy versus Transport Studies†

Biochemistry ◽  
2007 ◽  
Vol 46 (10) ◽  
pp. 2797-2804 ◽  
Author(s):  
Theeraporn Puntheeranurak ◽  
Barbara Wimmer ◽  
Francisco Castaneda ◽  
Hermann J. Gruber ◽  
Peter Hinterdorfer ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Substrate Specificity ◽  
Single Molecule ◽  
Sugar Transport ◽  
Force Microscopy ◽  
Atomic Force ◽  
Transport Studies

Going Vertical To Improve the Accuracy of Atomic Force Microscopy Based Single-Molecule Force Spectroscopy

ACS Nano ◽  
2017 ◽  
Vol 12 (1) ◽  
pp. 198-207 ◽  
Author(s):  
Robert Walder ◽  
William J. Van Patten ◽  
Ayush Adhikari ◽  
Thomas T. Perkins
Keyword(s):  
Atomic Force Microscopy ◽  
Single Molecule ◽  
Force Spectroscopy ◽  
Single Molecule Force Spectroscopy ◽  
Force Microscopy ◽  
Atomic Force

Investigation of Perylene Photonic Wires by Combined Single-Molecule Fluorescence and Atomic Force Microscopy

2004 ◽  
Vol 116 (31) ◽  
pp. 4137-4141 ◽  
Author(s):  
Jordi Hernando ◽  
Pieter A. J. de Witte ◽  
Erik M. H.P van Dijk ◽  
Jeroen Korterik ◽  
Roeland J. M. Nolte ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Single Molecule ◽  
Single Molecule Fluorescence ◽  
Force Microscopy ◽  
Atomic Force ◽  
Photonic Wires
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