scholarly journals Fluorescence resonance energy transfer and protein-induced fluorescence enhancement as synergetic multi-scale molecular rulers

2016 ◽  
Author(s):  
Evelyn Ploetz ◽  
Eitan Lerner ◽  
Florence Husada ◽  
Martin Roelfs ◽  
SangYoon Chung ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Single Molecule ◽  
Conformational Changes ◽  
Transcription Initiation ◽  
Structural Changes ◽  
Fluorescence Enhancement ◽  
Protein Complexes ◽  
Fundamental Problem ◽  
Resonance Energy Transfer ◽  
Resonance Energy

ABSTRACTAdvanced microscopy methods allow obtaining information on (dynamic) conformational changes in biomolecules via measuring a single molecular distance in the structure. It is, however, extremely challenging to capture the full depth of a three-dimensional biochemical state, binding-related structural changes or conformational cross-talk in multi-protein complexes using one-dimensional assays. In this paper we address this fundamental problem by extending the standard molecular ruler based on Förster resonance energy transfer (FRET) into a two-dimensional assay via its combination with protein-induced fluorescence enhancement (PIFE). We show that donor brightness (viaPIFE) and energy transfer efficiency (viaFRET) can simultaneously report on e.g., the conformational state of dsDNA following its interaction with unlabelled proteins (BamHI, EcoRV, T7 DNA polymerase gp5/trx). The PIFE-FRET assay uses established labelling protocols and single molecule fluorescence detection schemes (alternating-laser excitation, ALEX). Besides quantitative studies of PIFE and FRET ruler characteristics, we outline possible applications of ALEX-based PIFE-FRET for single-molecule studies with diffusing and immobilized molecules. Finally, we study transcription initiation and scrunching ofE. coliRNA-polymerase with PIFE-FRET and provide direct evidence for the physical presence and vicinity of the polymerase that causes structural changes and scrunching of the transcriptional DNA bubble.

scholarly journals Transcription initiation at a consensus bacterial promoter proceeds via a 'bind-unwind-load-and-lock' mechanism

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Abhishek Mazumder ◽  
Richard H Ebright ◽  
Achillefs Kapanidis
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Transcription Initiation ◽  
Core Promoter ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Extensive Study ◽  
Active Centre ◽  
Transient Nature ◽  
Promoter Dna

Transcription initiation starts with unwinding of promoter DNA by RNA polymerase (RNAP) to form a catalytically competent RNAP-promoter complex (RPO). Despite extensive study, the mechanism of promoter unwinding has remained unclear, in part due to the transient nature of intermediates on path to RPo. Here, using single-molecule unwinding-induced fluorescence enhancement to monitor promoter unwinding, and single-molecule fluorescence resonance energy transfer to monitor RNAP clamp conformation, we analyze RPo formation at a consensus bacterial core promoter. We find that the RNAP clamp is closed during promoter binding, remains closed during promoter unwinding, and then closes further, locking the unwound DNA in the RNAP active-centre cleft. Our work defines a new, 'bind-unwind-load-and-lock' model for the series of conformational changes occurring during promoter unwinding at a consensus bacterial promoter and provides the tools needed to examine the process in other organisms and at other promoters.

Single-molecule fluorescence resonance energy transfer techniques on rotary ATP synthases

2011 ◽  
Vol 392 (1-2) ◽  
Author(s):  
Michael Börsch
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Single Molecule ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Single Molecule Fret ◽  
Intensity Changes ◽  
Fluorescence Resonance

Abstract Conformational changes of proteins can be monitored in real time by fluorescence resonance energy transfer (FRET). Two different fluorophores have to be attached to those protein domains which move during function. Distance fluctuations between the fluorophores are measured by relative fluorescence intensity changes or fluorescence lifetime changes. The rotary mechanics of the two motors of FoF1-ATP synthase have been studied in vitro by single-molecule FRET. The results are summarized and perspectives for other transport ATPases are discussed.

scholarly journals Cooperative Analysis of Structural Dynamics in RNA-Protein Complexes by Single-Molecule Förster Resonance Energy Transfer Spectroscopy

Molecules ◽  
2020 ◽  
Vol 25 (9) ◽  
pp. 2057 ◽  
Author(s):  
Nathalie Meiser ◽  
Christin Fuks ◽  
Martin Hengesbach
Keyword(s):  
Energy Transfer ◽  
Single Molecule ◽  
Protein Complexes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Forster Resonance Energy Transfer ◽  
Telomere Maintenance ◽  
Förster Resonance Energy Transfer ◽  
Rna Protein Complexes ◽  
Förster Resonance

RNA-protein complexes (RNPs) are essential components in a variety of cellular processes, and oftentimes exhibit complex structures and show mechanisms that are highly dynamic in conformation and structure. However, biochemical and structural biology approaches are mostly not able to fully elucidate the structurally and especially conformationally dynamic and heterogeneous nature of these RNPs, to which end single molecule Förster resonance energy transfer (smFRET) spectroscopy can be harnessed to fill this gap. Here we summarize the advantages of strategic smFRET studies to investigate RNP dynamics, complemented by structural and biochemical data. Focusing on recent smFRET studies of three essential biological systems, we demonstrate that investigation of RNPs on a single molecule level can answer important functional questions that remained elusive with structural or biochemical approaches alone: The complex structural rearrangements throughout the splicing cycle, unwinding dynamics of the G-quadruplex (G4) helicase RHAU, and aspects in telomere maintenance regulation and synthesis.

scholarly journals Two-colour single-molecule photoinduced electron transfer fluorescence imaging microscopy of chaperone dynamics

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jonathan Schubert ◽  
Andrea Schulze ◽  
Chrisostomos Prodromou ◽  
Hannes Neuweiler
Keyword(s):  
Electron Transfer ◽  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Conformational Changes ◽  
Photoinduced Electron Transfer ◽  
Structural Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescence Imaging Microscopy ◽  
Pet Probes

AbstractMany proteins are molecular machines, whose function is dependent on multiple conformational changes that are initiated and tightly controlled through biochemical stimuli. Their mechanistic understanding calls for spectroscopy that can probe simultaneously such structural coordinates. Here we present two-colour fluorescence microscopy in combination with photoinduced electron transfer (PET) probes as a method that simultaneously detects two structural coordinates in single protein molecules, one colour per coordinate. This contrasts with the commonly applied resonance energy transfer (FRET) technique that requires two colours per coordinate. We demonstrate the technique by directly and simultaneously observing three critical structural changes within the Hsp90 molecular chaperone machinery. Our results reveal synchronicity of conformational motions at remote sites during ATPase-driven closure of the Hsp90 molecular clamp, providing evidence for a cooperativity mechanism in the chaperone’s catalytic cycle. Single-molecule PET fluorescence microscopy opens up avenues in the multi-dimensional exploration of protein dynamics and allosteric mechanisms.

scholarly journals Quantitative single-molecule three-color Förster resonance energy transfer by photon distribution analysis

2018 ◽  
Author(s):  
Anders Barth ◽  
Lena Voith von Voithenberg ◽  
Don C. Lamb
Keyword(s):  
Energy Transfer ◽  
Single Molecule ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Forster Resonance Energy Transfer ◽  
Förster Resonance Energy Transfer ◽  
Distribution Analysis ◽  
Photon Distribution ◽  
Förster Resonance

AbstractSingle-molecule Förster resonance energy transfer (FRET) is a powerful tool to study conformational dynamics of biomolecules. Using solution-based single-pair FRET by burst analysis, conformational heterogeneities and fluctuations of fluorescently labeled proteins or nucleic acids can be studied by monitoring a single distance at a time. Three-color FRET is sensitive to three distances simultaneously and can thus elucidate complex coordinated motions within single molecules. While three-color FRET has been applied on the single-molecule level before, a detailed quantitative description of the obtained FRET efficiency distributions is still missing. Direct interpretation of three-color FRET data is additionally complicated by an increased shot noise contribution when converting photon counts to FRET efficiencies. However, to address the question of coordinated motion, it is of special interest to extract information about the underlying distance heterogeneity, which is not easily extracted from the FRET efficiency histograms directly. Here, we present three-color photon distribution analysis (3C-PDA), a method to extract distributions of inter-dye distances from three-color FRET measurements. We present a model for diffusion-based three-color FRET experiments and apply Bayesian inference to extract information about the physically relevant distance heterogeneity in the sample. The approach is verified using simulated data sets and experimentally applied to triple-labeled DNA duplexes. Finally, 3C-FRET experiments on the Hsp70 chaperone BiP reveal conformational coordinated changes between individual domains. The possibility to address the co-occurrence of intramolecular distances makes 3C-PDA a powerful method to study the coordination of domain motions within biomolecules during conformational changes.SignificanceIn solution-based single-molecule Förster resonance energy transfer (FRET) experiments, biomolecules are studied as they freely diffuse through the observation volume of a confocal microscope, resulting in bursts of fluorescence from single molecules. Using three fluorescent labels, one can concurrently measure three distances in a single molecule but the experimentally limited number of photons is not sufficient for a straight-forward analysis. Here, we present a probabilistic framework, called three-color photon distribution analysis (3C-PDA), to extract quantitative information from single-molecule three-color FRET experiments. By extracting distributions of interdye distances from the data, the method provides a three-dimensional description of the conformational space of biomolecules, enabling the detection of coordinated movements during conformational changes.

Combining gold nanoparticle antennas with single-molecule fluorescence resonance energy transfer (smFRET) to study DNA hairpin dynamics

Nanoscale ◽  
2018 ◽  
Vol 10 (14) ◽  
pp. 6611-6619 ◽  
Author(s):  
Jinyong Hu ◽  
Meiyan Wu ◽  
Li Jiang ◽  
Zhensheng Zhong ◽  
Zhangkai Zhou ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Gold Nanoparticle ◽  
Fluorescence Resonance Energy Transfer ◽  
Single Molecule ◽  
Fluorescence Enhancement ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dna Hairpin ◽  
Single Molecule Fluorescence ◽  
Fluorescence Resonance

Gold nanoparticle antennas as a promising platform not only for fluorescence enhancement but also for the studies of single-molecule kinetics.

Subunit movement in individual H+-ATP synthases during ATP synthesis and hydrolysis revealed by fluorescence resonance energy transfer

2005 ◽  
Vol 33 (4) ◽  
pp. 878-882 ◽  
Author(s):  
M. Börsch ◽  
P. Gräber
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Single Molecule ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Atp Synthesis ◽  
Fluorescence Resonance ◽  
Atp Synthases

F-type H+-ATP synthases synthesize ATP from ADP and phosphate using the energy supplied by a transmembrane electrochemical potential difference of protons. Rotary subunit movements within the enzyme drive catalysis in either an ATP hydrolysis or an ATP synthesis direction respectively. To monitor these subunit movements and associated conformational changes in real time and with subnanometre resolution, a single-molecule FRET (fluorescence resonance energy transfer) approach has been developed using the double-labelled H+-ATP synthase from Escherichia coli. After reconstitution into a liposome, this enzyme was able to catalyse ATP synthesis when the membrane was energized.

scholarly journals Transcription initiation at a consensus bacterial promoter proceeds via a "bind-unwind-load-and-lock" mechanism

2021 ◽  
Author(s):  
Abhishek Mazumder ◽  
Richard H Ebright ◽  
Achillefs Kapanidis
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Transcription Initiation ◽  
Core Promoter ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Extensive Study ◽  
Active Centre ◽  
Transient Nature ◽  
Promoter Dna

Transcription initiation starts with unwinding of promoter DNA by RNA polymerase (RNAP) to form a catalytically competent RNAP-promoter complex (RPO). Despite extensive study, the mechanism of promoter unwinding has remained unclear, in part due to the transient nature of intermediates on path to RPo. Here, using single-molecule unwinding-induced fluorescence enhancement to monitor promoter unwinding, and single-molecule fluorescence resonance energy transfer to monitor RNAP clamp conformation, we analyze RPo formation at a consensus bacterial core promoter. We find that the RNAP clamp is closed during promoter binding, remains closed during promoter unwinding, and then closes further, locking the unwound DNA in the RNAP active-centre cleft. Our work defines a new, bind-unwind-load-and-lock, model for the series of conformational changes occurring during promoter unwinding at a consensus bacterial promoter and provides the tools needed to examine the process in other organisms and at other promoters.

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