High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

Ma, Junyan; Yanez-Orozco, Inna S.; Adariani, Soheila Rezaei; Dolino, Drew M.; Jayaraman, Vasanthi; Sanabria, Hugo

doi:10.3791/55623

Cited by 17 publications

(15 citation statements)

References 65 publications

(72 reference statements)

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“…A home-built confocal system was used for the MFS-smFRET measurements and calibration, as described in detail elsewhere (Ma et al 2017;Hellenkamp et al 2018;Yanez Orozco et al 2018;Hamilton and Sanabria 2019).…”

Section: Multiparameter Fluorescence Spectroscopy (Mfs) For Single-molecule Fret Experiments (Smfret)mentioning

confidence: 99%

Ensemble switching unveils a kinetic rheostat mechanism of the eukaryotic thiamine pyrophosphate riboswitch

Saikia

Godar

et al. 2021

RNA

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Thiamine pyrophosphate (TPP) riboswitches regulate thiamine metabolism by inhibiting the translation of enzymes essential to thiamine synthesis pathways upon binding to thiamine pyrophosphate in cells across all domains of life. Recent work on the Arabidopsis thaliana TPP riboswitch suggests a multi-step TPP binding process involving multiple riboswitch configurational ensembles and Mg 2+ dependence underlies the mechanism of TPP recognition and subsequent transition to the expression-inhibiting state of the aptamer domain followed by changes in the expression platform. However, details of the relationship between TPP riboswitch conformational changes and interactions with TPP and Mg 2+ in the aptamer domain constituting this mechanism are unknown. Therefore, we integrated single-molecule multiparameter fluorescence and force spectroscopy with atomistic molecular dynamics simulations and found that conformational transitions within the aptamer domain's sensor helices associated with TPP and Mg 2+ ligand binding occurred between at least five different ensembles on timescales ranging from µs to ms. These dynamics are orders of magnitude faster than the ten second-timescale folding kinetics associated with expression-state switching in the switch helix. Together, our results show that a TPP and Mg 2+ dependent mechanism determines dynamic configurational state ensemble switching of the aptamer domain's sensor helices that regulate the switch helix's stability, which ultimately may lead to the expressioninhibiting state of the riboswitch. Additionally, we propose that two pathways exist for ligand recognition and that this mechanism underlies a kinetic rheostat-like behavior of the Arabidopsis thaliana TPP riboswitch.

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Section: Multiparameter Fluorescence Spectroscopy (Mfs) For Single-molecule Fret Experiments (Smfret)mentioning

confidence: 99%

Ensemble switching unveils a kinetic rheostat mechanism of the eukaryotic thiamine pyrophosphate riboswitch

Saikia

Godar

et al. 2021

RNA

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“…This involves monitoring the time-dependent structural changes in a single protein molecule that is labeled with a pair of donor and acceptor molecules at the site of interest (Weiss, 2000; Roy et al, 2008). Typically, the fluorophores used in smFRET studies have R o of ~50 Å (Ma et al, 2017). The advantage of smFRET over ensemble FRET is that it can detect conformational dynamics and heterogeneity within a single protein molecule instead of measuring average interactions among protein molecules in an ensemble.…”

Section: Site-directed Fluorescence (Sdfl) Approachesmentioning

confidence: 99%

Site-Directed Fluorescence Approaches for Dynamic Structural Biology of Membrane Peptides and Proteins

Raghuraman

Chatterjee

Das

2019

Front. Mol. Biosci.

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Membrane proteins mediate a number of cellular functions and are associated with several diseases and also play a crucial role in pathogenicity. Due to their importance in cellular structure and function, they are important drug targets for ~60% of drugs available in the market. Despite the technological advancement and recent successful outcomes in determining the high-resolution structural snapshot of membrane proteins, the mechanistic details underlining the complex functionalities of membrane proteins is least understood. This is largely due to lack of structural dynamics information pertaining to different functional states of membrane proteins in a membrane environment. Fluorescence spectroscopy is a widely used technique in the analysis of functionally-relevant structure and dynamics of membrane protein. This review is focused on various site-directed fluorescence (SDFL) approaches and their applications to explore structural information, conformational changes, hydration dynamics, and lipid-protein interactions of important classes of membrane proteins that include the pore-forming peptides/proteins, ion channels/transporters and G-protein coupled receptors.

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“…The home-built confocal system used for MFS-smFRET and calibration was described in detail elsewhere (30,38–40). To briefly summarize, we use Pulsed Interleaved Excitation (PIE) (41), or Alternating Laser EXcitation (ALEX) (42), with two diode lasers (Model LDH-D-C- 485 at 485 nm and laser LDH-D-C-640 at 640 nm; PicoQuant, Germany) operating at 40 MHz with 25 ns interleaved time.…”

Section: Methodsmentioning

confidence: 99%

“…The data analysis used software developed in the Seidel group and is available at http://www.mpc.hhu.de/en/software (37,38,(43)(44)(45). MFS accounts for many challenges associated with smFRET experiments, including photo-bleaching, cis-trans acceptor blinking, and dynamic and static quenching.…”

Section: Multiparameter Fluorescence Spectroscopy (Mfs) For Single-momentioning

confidence: 99%

“…Although the shape of the distribution is less important, the mean and the variance are both relevant statistical parameters in single-molecule FRET experiments (48). Therefore, we fixed the variance according to benchmark studies (37)(38)(39)44) and optimized for the mean. Next, we evaluated the best results by visual inspection of the weighted residuals, monitoring the distribution of the autocorrelation of the residuals, and by a statistical improvement of the figure of merit, 2 (31,49) (SI Equation 7).…”

Section: Time-correlated Single Photon Counting (Tcspc) and Filtered mentioning

confidence: 99%

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Ensemble Switching Unveils a Kinetic Rheostat Mechanism of the Eukaryotic Thiamine Pyrophosphate Riboswitch

Saikia

Godar

et al. 2021

Preprint

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Thiamine pyrophosphate (TPP) riboswitches regulate thiamine metabolism by inhibiting the translation of enzymes essential to thiamine synthesis pathways upon binding to thiamine pyrophosphate in cells across all domains of life. Recent work on the Arabidopsis thaliana TPP riboswitch suggests a multi-step TPP binding process involving multiple riboswitch conformational ensembles and that Mg2+ dependence underlies the mechanism of TPP recognition and subsequent transition to the translation-inhibiting state of the switching sequence followed by changes in the expression platform. However, details of the relationship between TPP riboswitch conformational changes and interactions with TPP and Mg2+ in the aptamer domain constituting this mechanism are unknown. Therefore, we integrated single-molecule multiparameter fluorescence and force spectroscopy with atomistic molecular dynamics simulations and found that conformational transitions within the aptamer domain associated with TPP and Mg2+ ligand binding occurred between at least five different ensembles on timescales ranging from μs to ms. These dynamics are at least an order of magnitude faster than folding and unfolding kinetics associated with translation-state switching in the switching sequence. Moreover, we propose that two pathways exist for ligand recognition. Together, our results suggest a dynamic ensemble switching of the aptamer domain that may lead to the translation-inhibiting state of the riboswitch. Additionally, our results suggest that multiple configurations could enable inhibitory tuning manifested through ligand-dependent changes via ensemble switching and kinetic rheostat-like behavior of the Arabidopsis thaliana TPP riboswitch.

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High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

Cited by 17 publications

References 65 publications

Ensemble switching unveils a kinetic rheostat mechanism of the eukaryotic thiamine pyrophosphate riboswitch

Ensemble switching unveils a kinetic rheostat mechanism of the eukaryotic thiamine pyrophosphate riboswitch

Site-Directed Fluorescence Approaches for Dynamic Structural Biology of Membrane Peptides and Proteins

Ensemble Switching Unveils a Kinetic Rheostat Mechanism of the Eukaryotic Thiamine Pyrophosphate Riboswitch

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