Single-Molecule FRET: A Tool to Characterize DNA Nanostructures

Pal, Nibedita

doi:10.3389/fmolb.2022.835617

Cited by 3 publications

(3 citation statements)

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“…FRET has been applied in investigations of a large variety of objects and phenomena. In a series of studies, FRET was utilized to obtain otherwise difficult to observe structural information of various molecules in situ and in vivo (Feng, Poyton, & Ha, 2021; Gayrard & Borghi, 2016; Lerner et al., 2021; Pal, 2022; Terai, Imanishi, Li, & Matsuda, 2019; Voith von Voithenberg & Lamb, 2018). FRET has also been used to design high sensitivity sensors for various biological assays (Imani, Mohajeri, Rastegar, & Zarghami, 2021; Terai et al., 2019; Weihs, Anderson, Trowell, & Caron, 2021), and FRET has been extensively used in plant research (Duan, Li, Duan, Zhang, & Xing, 2022).…”

Section: Applicationsmentioning

confidence: 99%

“…Conformational changes in the 10 µs to 10 s range can be best studied using diffusion‐based smFRET (Tomov et al., 2012). With the established and improved smFRET schemes, several problems have been studied, such as the structural dynamics of ion channels (Martinac, 2017), membrane proteins (Castell, Dijkman, Wiseman, & Goddard, 2018; Ward, Ye, Schuster, Wei, & Barrera, 2021; Yang, Xu, Wang, Chen, & Zhao, 2021), intrinsically disordered proteins (LeBlanc, Kulkarni, & Weninger, 2018; Metskas & Rhoades, 2020), DNA polymerases (Millar, 2022), and DNA nanostructures (Pal, 2022). The multicolor version of smFRET, in which more than one donor‐acceptor pair is used, can provide even more information about protein‐DNA interactions, chromatin remodeling and even protein translation (Feng et al., 2021).…”

Section: Applicationsmentioning

confidence: 99%

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Principles of Resonance Energy Transfer

2022

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This unit describes the basic principles of Förster resonance energy transfer (FRET). Beginning with a brief summary of the history of FRET applications, the theory of FRET is introduced in detail using figures to explain all the important parameters of the FRET process. After listing various approaches for measuring FRET efficiency, several pieces of advice are given on choosing the appropriate instrumentation. The unit concludes with a discussion of the limitations of FRET measurements followed by a few examples of the latest FRET applications, including new developments such as spectral flow cytometric FRET, single-molecule FRET, and combinations of FRET with super-resolution or lifetime imaging microscopy and with molecular dynamics simulations.

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Section: Applicationsmentioning

confidence: 99%

Section: Applicationsmentioning

confidence: 99%

Principles of Resonance Energy Transfer

2022

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“…Alternatively, the sample may be imaged cooled down (as we did for mammalian enzyme [ 25 ]) or with very limiting substrate concentrations to slow down turnover. Another approach could be to label appropriate residues on the surface of PA and MA so that single-molecule FRET signals [ 58 ] can be recorded and compared in the absence and presence of turnover. Finally, time-resolved cryo-EM methods are now approaching ∼5 ms resolution [ 59 , 60 ] and could be applied to visualise first steps in complex I reaction after addition of substrates.…”

Section: Remaining Questions and Future Workmentioning

confidence: 99%

From the ‘black box' to ‘domino effect' mechanism: what have we learned from the structures of respiratory complex I

Sazanov

2023

Biochemical Journal

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My group and myself have studied respiratory complex I for almost 30 years, starting in 1994 when it was known as a L-shaped giant ‘black box' of bioenergetics. First breakthrough was the X-ray structure of the peripheral arm, followed by structures of the membrane arm and finally the entire complex from Thermus thermophilus. The developments in cryo-EM technology allowed us to solve the first complete structure of the twice larger, ∼1 MDa mammalian enzyme in 2016. However, the mechanism coupling, over large distances, the transfer of two electrons to pumping of four protons across the membrane remained an enigma. Recently we have solved high-resolution structures of mammalian and bacterial complex I under a range of redox conditions, including catalytic turnover. This allowed us to propose a robust and universal mechanism for complex I and related protein families. Redox reactions initially drive conformational changes around the quinone cavity and a long-distance transfer of substrate protons. These set up a stage for a series of electrostatically driven proton transfers along the membrane arm (‘domino effect'), eventually resulting in proton expulsion from the distal antiporter-like subunit. The mechanism radically differs from previous suggestions, however, it naturally explains all the unusual structural features of complex I. In this review I discuss the state of knowledge on complex I, including the current most controversial issues.

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