Observation of long-range tertiary interactions during ligand binding by the TPP riboswitch aptamer

The thiamine pyrophosphate (TPP) riboswitch is a cis-regulatory element in mRNA that modifies gene expression in response to TPP concentration. Its specificity is dependent upon conformational changes that take place within its aptamer domain. Here, the role of tertiary interactions in ligand binding was studied at the single-molecule level by combined force spectroscopy and Förster resonance energy transfer (smFRET), using an optical trap equipped for simultaneous smFRET. The ‘Force-FRET’ approach directly probes secondary and tertiary structural changes during folding, including events associated with binding. Concurrent transitions observed in smFRET signals and RNA extension revealed differences in helix-arm orientation between two previously-identified ligand-binding states that had been undetectable by spectroscopy alone. Our results show that the weaker binding state is able to bind to TPP, but is unable to form a tertiary docking interaction that completes the binding process. Long-range tertiary interactions stabilize global riboswitch structure and confer increased ligand specificity.

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

Nature Communications ◽

10.1038/s41467-021-27286-5 ◽

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.

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

10.1101/047779 ◽

2016 ◽

Cited By ~ 1

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.

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Interbase-FRET binding assay for pre-microRNAs

Scientific Reports ◽

10.1038/s41598-021-88922-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Mattias Bood ◽

Anna Wypijewska del Nogal ◽

Jesper R. Nilsson ◽

Fredrik Edfeldt ◽

Anders Dahlén ◽

...

Keyword(s):

Ligand Binding ◽

Drug Targets ◽

Structural Changes ◽

Small Sample Size ◽

Resonance Energy ◽

Small Sample ◽

Titration Calorimetry ◽

Drug Candidate ◽

Binding Studies ◽

Aberrant Expression

AbstractThe aberrant expression of microRNAs (miRs) has been linked to several human diseases. A promising approach for targeting these anomalies is the use of small-molecule inhibitors of miR biogenesis. These inhibitors have the potential to (i) dissect miR mechanisms of action, (ii) discover new drug targets, and (iii) function as new therapeutic agents. Here, we designed Förster resonance energy transfer (FRET)-labeled oligoribonucleotides of the precursor of the oncogenic miR-21 (pre-miR-21) and used them together with a set of aminoglycosides to develop an interbase-FRET assay to detect ligand binding to pre-miRs. Our interbase-FRET assay accurately reports structural changes of the RNA oligonucleotide induced by ligand binding. We demonstrate its application in a rapid, qualitative drug candidate screen by assessing the relative binding affinity between 12 aminoglycoside antibiotics and pre-miR-21. Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) were used to validate our new FRET method, and the accuracy of our FRET assay was shown to be similar to the established techniques. With its advantages over SPR and ITC owing to its high sensitivity, small sample size, straightforward technique and the possibility for high-throughput expansion, we envision that our solution-based method can be applied in pre-miRNA–target binding studies.

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

eLife ◽

10.7554/elife.70090 ◽

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.

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Comparative analysis of the coordinated motion of Hsp70s from different organelles observed by single-molecule three-color FRET

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2025578118 ◽

2021 ◽

Vol 118 (33) ◽

pp. e2025578118

Author(s):

Lena Voith von Voithenberg ◽

Anders Barth ◽

Vanessa Trauschke ◽

Benjamin Demarco ◽

Swati Tyagi ◽

...

Keyword(s):

Single Molecule ◽

Structural Changes ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Conformational Space ◽

Distribution Analysis ◽

Nucleotide Exchange ◽

Nucleotide Analogs ◽

Recognition Mechanism ◽

Substrate Peptide

Cellular function depends on the correct folding of proteins inside the cell. Heat-shock proteins 70 (Hsp70s), being among the first molecular chaperones binding to nascently translated proteins, aid in protein folding and transport. They undergo large, coordinated intra- and interdomain structural rearrangements mediated by allosteric interactions. Here, we applied a three-color single-molecule Förster resonance energy transfer (FRET) combined with three-color photon distribution analysis to compare the conformational cycle of the Hsp70 chaperones DnaK, Ssc1, and BiP. By capturing three distances simultaneously, we can identify coordinated structural changes during the functional cycle. Besides the known conformations of the Hsp70s with docked domains and open lid and undocked domains with closed lid, we observed additional intermediate conformations and distance broadening, suggesting flexibility of the Hsp70s in adopting the states in a coordinated fashion. Interestingly, the difference of this distance broadening varied between DnaK, Ssc1, and BiP. Study of their conformational cycle in the presence of substrate peptide and nucleotide exchange factors strengthened the observation of additional conformational intermediates, with BiP showing coordinated changes more clearly compared to DnaK and Ssc1. Additionally, DnaK and BiP were found to differ in their selectivity for nucleotide analogs, suggesting variability in the recognition mechanism of their nucleotide-binding domains for the different nucleotides. By using three-color FRET, we overcome the limitations of the usual single-distance approach in single-molecule FRET, allowing us to characterize the conformational space of proteins in higher detail.

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Identification of Allosteric Fingerprints of Alpha-Synuclein Aggregates in Matrix Metalloprotease-1 and Substrate-Specific Virtual Screening with Single Molecule Insights

10.21203/rs.3.rs-1132741/v1 ◽

2021 ◽

Author(s):

Sumaer Kamboj ◽

Chase Harms ◽

Derek Wright ◽

Anthony Nash ◽

Lokender Kumar ◽

...

Keyword(s):

Virtual Screening ◽

Single Molecule ◽

Conformational Changes ◽

Single Point ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Alpha Synuclein ◽

Matrix Metalloproteases ◽

Single Point Mutation ◽

Mmp Inhibitor

Abstract Alpha-synuclein (aSyn) has implications in pathological protein aggregations in neurodegeneration. Matrix metalloproteases (MMPs) are broad-spectrum proteases and cleave aSyn, leading to aggregation. Previously, we showed that allosteric communications between the two domains of MMP1 on collagen fibril and fibrin depend on substrates, activity, and ligands. Here we report quantification of allostery using single molecule measurements of MMP1 dynamics on aSyn-induced aggregates by calculating Forster Resonance Energy Transfer (FRET) between two dyes attached to the catalytic and hemopexin domains of MMP1. The two domains of MMP1 prefer open conformations that are inhibited by a single point mutation E219Q of MMP1 and tetracycline, an MMP inhibitor. A two-state Poisson process describes the interdomain dynamics, where the two states and kinetic rates of interconversion between them are obtained from histograms and autocorrelations of FRET values. Since a crystal structure of aSyn-bound MMP1 is not available, we performed molecular docking of MMP1 with aSyn using ClusPro. We simulated MMP1 dynamics using different docking poses and matched the experimental and simulated interdomain dynamics to identify an appropriate pose. We used experimentally validated simulations to define conformational changes at the catalytic site and identify allosteric residues in the hemopexin domain having strong correlations with the catalytic motif residues. We defined Shannon entropy to quantify MMP1 dynamics. We performed virtual screening against a site on selected aSyn-MMP1 binding poses and showed that lead molecules differ between free MMP1 and substrate-bound MMP1. Also, identifying aSyn-specific allosteric residues in MMP1 enabled further selection of lead molecules. In other words, virtual screening needs to take substrates into account for substrate-specific control of MMP1 activity. Molecular understanding of interactions between MMP1 and aSyn-induced aggregates may open up the possibility of degrading aggregates by targeting MMPs.

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Extreme mechanical diversity of human telomeric DNA revealed by fluorescence-force spectroscopy

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1815162116 ◽

2019 ◽

Vol 116 (17) ◽

pp. 8350-8359 ◽

Cited By ~ 19

Author(s):

Jaba Mitra ◽

Monika A. Makurath ◽

Thuy T. M. Ngo ◽

Alice Troitskaia ◽

Yann R. Chemla ◽

...

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Conformational Changes ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Force Spectroscopy ◽

Telomeric Dna ◽

Telomeric Sequences ◽

Substitution Mutant

G-quadruplexes (GQs) can adopt diverse structures and are functionally implicated in transcription, replication, translation, and maintenance of telomere. Their conformational diversity under physiological levels of mechanical stress, however, is poorly understood. We used single-molecule fluorescence-force spectroscopy that combines fluorescence resonance energy transfer with optical tweezers to measure human telomeric sequences under tension. Abrupt GQ unfolding with K+in solution occurred at as many as four discrete levels of force. Added to an ultrastable state and a gradually unfolding state, there were six mechanically distinct structures. Extreme mechanical diversity was also observed with Na+, although GQs were mechanically weaker. Our ability to detect small conformational changes at low forces enabled the determination of refolding forces of about 2 pN. Refolding was rapid and stochastically redistributed molecules to mechanically distinct states. A single guanine-to-thymine substitution mutant required much higher ion concentrations to display GQ-like unfolding and refolded via intermediates, contrary to the wild type. Contradicting an earlier proposal, truncation to three hexanucleotide repeats resulted in a single-stranded DNA-like mechanical behavior under all conditions, indicating that at least four repeats are required to form mechanically stable structures.

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Irreversible chemical steps control intersubunit dynamics during translation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0805299105 ◽

2008 ◽

Vol 105 (40) ◽

pp. 15364-15369 ◽

Cited By ~ 82

Author(s):

R. Andrew Marshall ◽

Magdalena Dorywalska ◽

Joseph D. Puglisi

Keyword(s):

Single Molecule ◽

Conformational Changes ◽

Large Scale ◽

Bond Formation ◽

Elongation Factor ◽

Resonance Energy ◽

Peptide Bond ◽

Peptide Bond Formation ◽

Gtp Hydrolysis ◽

30S Subunit

The ribosome, a two-subunit macromolecular machine, deciphers the genetic code and catalyzes peptide bond formation. Dynamic rotational movement between ribosomal subunits is likely required for efficient and accurate protein synthesis, but direct observation of intersubunit dynamics has been obscured by the repetitive, multistep nature of translation. Here, we report a collection of single-molecule fluorescence resonance energy transfer assays that reveal a ribosomal intersubunit conformational cycle in real time during initiation and the first round of elongation. After subunit joining and delivery of correct aminoacyl-tRNA to the ribosome, peptide bond formation results in a rapid conformational change, consistent with the counterclockwise rotation of the 30S subunit with respect to the 50S subunit implied by prior structural and biochemical studies. Subsequent binding of elongation factor G and GTP hydrolysis results in a clockwise rotation of the 30S subunit relative to the 50S subunit, preparing the ribosome for the next round of tRNA selection and peptide bond formation. The ribosome thus harnesses the free energy of irreversible peptidyl transfer and GTP hydrolysis to surmount activation barriers to large-scale conformational changes during translation. Intersubunit rotation is likely a requirement for the concerted movement of tRNA and mRNA substrates during translocation.

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Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ

Open Biology ◽

10.1098/rsob.200406 ◽

2021 ◽

Vol 11 (4) ◽

Author(s):

Evelyn Ploetz ◽

Gea K. Schuurman-Wolters ◽

Niels Zijlstra ◽

Amarins W. Jager ◽

Douglas A. Griffith ◽

...

Keyword(s):

Ligand Binding ◽

Single Molecule ◽

Conformational Changes ◽

Protein Interactions ◽

Transmembrane Domain ◽

Substrate Binding ◽

Titration Calorimetry ◽

Uptake System ◽

Conformational States ◽

Binding Domains

The ATP-binding cassette transporter GlnPQ is an essential uptake system that transports glutamine, glutamic acid and asparagine in Gram-positive bacteria. It features two extra-cytoplasmic substrate-binding domains (SBDs) that are linked in tandem to the transmembrane domain of the transporter. The two SBDs differ in their ligand specificities, binding affinities and their distance to the transmembrane domain. Here, we elucidate the effects of the tandem arrangement of the domains on the biochemical, biophysical and structural properties of the protein. For this, we determined the crystal structure of the ligand-free tandem SBD1-2 protein from Lactococcus lactis in the absence of the transporter and compared the tandem to the isolated SBDs. We also used isothermal titration calorimetry to determine the ligand-binding affinity of the SBDs and single-molecule Förster resonance energy transfer (smFRET) to relate ligand binding to conformational changes in each of the domains of the tandem. We show that substrate binding and conformational changes are not notably affected by the presence of the adjoining domain in the wild-type protein, and changes only occur when the linker between the domains is shortened. In a proof-of-concept experiment, we combine smFRET with protein-induced fluorescence enhancement (PIFE–FRET) and show that a decrease in SBD linker length is observed as a linear increase in donor-brightness for SBD2 while we can still monitor the conformational states (open/closed) of SBD1. These results demonstrate the feasibility of PIFE–FRET to monitor protein–protein interactions and conformational states simultaneously.

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Conformational Dynamics Related to Membrane Fusion Observed in Single Ebola GP Molecules

Proceedings ◽

10.3390/proceedings2020050049 ◽

2020 ◽

Vol 50 (1) ◽

pp. 49

Author(s):

Dibyendu Kumar Das ◽

Uriel Bulow ◽

Natasha D. Durham ◽

Ramesh Govindan ◽

James B. Munro

Keyword(s):

Membrane Fusion ◽

Single Molecule ◽

Conformational Changes ◽

Ebola Virus ◽

Conformational Dynamics ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Acidic Ph ◽

Cellular Environment ◽

Niemann Pick

The Ebola virus (EBOV) envelope glycoprotein (GP) is a membrane fusion machine required for virus entry into cells. Following the endocytosis of EBOV, the GP1 domain is cleaved by cellular cathepsins in acidic endosomes, exposing a binding site for the Niemann-Pick C1 (NPC1) receptor. The NPC1 binding to the cleaved GP1 is required for entry, but how this interaction translates to the GP2 domain-mediated fusion of viral and endosomal membranes is not known. Here, using a virus-liposome hemifusion assay and single-molecule Förster resonance energy transfer (smFRET)-imaging, we found that acidic pH, Ca2+, and NPC1 binding act synergistically to induce conformational changes in GP2 that drive lipid mixing. Acidic pH and Ca2+ shift the GP2 conformational equilibrium in favor of an intermediate state primed for NPC1 binding. GP1 cleavage and NPC1 binding enable GP2 to transition from a reversible intermediate to an irreversible conformation, suggestive of the post-fusion 6-helix bundle. Thus, the GP senses the cellular environment to protect against triggering prior to the arrival of EBOV in a permissive cellular compartment.

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