scholarly journals ATPase and protease domain movements in the bacterial AAA+ protease FtsH are driven by thermal fluctuations

2018 ◽  
Author(s):  
Martine Ruer ◽  
Georg Krainer ◽  
Philip Gröger ◽  
Michael Schlierf
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Thermotoga Maritima ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Thermal Fluctuations ◽  
Conformational Switching ◽  
Occupancy Distribution ◽  
Cellular Pathways ◽  
Domain Motions

AbstractAAA+ proteases are essential players in cellular pathways of protein degradation. Elucidating their conformational behavior is key for understanding their reaction mechanism and, importantly, for elaborating our understanding of mutation-induced protease deficiencies. Here, we study the structural dynamics of the Thermotoga maritima AAA+ hexameric ring metalloprotease FtsH (TmFtsH). Using a single-molecule Förster resonance energy transfer approach to monitor ATPase and protease inter-domain conformational changes in real time, we show that TmFtsH—even in the absence of nucleotide—is a highly dynamic protease undergoing conformational transitions between five states on the second timescale in a sequential manner. Addition of ATP does not influence the number of states nor change the timescale of domain motions, but affects the state occupancy distribution leading to an inter-domain compaction. These findings suggest that thermal energy, but not chemical energy, provides the major driving force for conformational switching, while ATP, through a state reequilibration, introduces directionality into this process. The TmFtsH A359V mutation, a homolog of the human pathogenic A510V mutation of paraplegin (SPG7) causing hereditary spastic paraplegia (HSP), does not affect the dynamic behavior of the protease but impairs the ATP-coupled domain compaction and, thus, may account for protease malfunctioning and pathogenesis in HSP.

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.

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

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.

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

2019 ◽  
Vol 116 (17) ◽  
pp. 8350-8359 ◽  
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.

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

Proceedings ◽  
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.

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 Single-molecule FRET dynamics of molecular motors in an ABEL Trap

2020 ◽  
Author(s):  
Maria Dienerowitz ◽  
Jamieson A. L. Howard ◽  
Steven D. Quinn ◽  
Frank Dienerowitz ◽  
Mark C. Leake
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Conformational Changes ◽  
Molecular Motors ◽  
Rational Design ◽  
Molecular Motor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Observation Time

AbstractSingle-molecule Förster resonance energy transfer (smFRET) of molecular motors provides transformative insights into their dynamics and conformational changes both at high temporal and spatial resolution simultaneously. However, a key challenge of such FRET investigations is to observe a molecule in action for long enough without restricting its natural function. The Anti-Brownian ELectrokinetic Trap (ABEL trap) sets out to combine smFRET with molecular confinement to enable observation times of up to several seconds while removing any requirement of tethered surface attachment of the molecule in question. In addition, the ABEL trap’s inherent ability to selectively capture FRET active molecules accelerates the data acquisition process. Here we exemplify the capabilities of the ABEL trap in performing extended timescale smFRET measurements on the molecular motor Rep, which is crucial for removing protein blocks ahead of the advancing DNA replication machinery and for restarting stalled DNA replication. We are able to monitor single Rep molecules up to 6 s with 1 ms time resolution capturing multiple conformational switching events during the observation time. Here we provide a step-by-step guide for the rational design, construction and implementation of the ABEL trap for smFRET detection of Rep in vitro. We include details of how to model the electric potential at the trap site and use Hidden Markov analysis of the smFRET trajectories.

scholarly journals Potent inhibitors of toxic alpha-synuclein identified via cellular time-resolved FRET biosensors

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Anthony R. Braun ◽  
Elly E. Liao ◽  
Mian Horvath ◽  
Prakriti Kalra ◽  
Karen Acosta ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Single Molecule ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Alpha Synuclein ◽  
Time Resolved ◽  
Primary Mouse ◽  
Fret Biosensors

AbstractWe have developed a high-throughput drug discovery platform, measuring fluorescence resonance energy transfer (FRET) with fluorescent alpha-synuclein (αSN) biosensors, to detect spontaneous pre-fibrillar oligomers in living cells. Our two αSN FRET biosensors provide complementary insight into αSN oligomerization and conformation in order to improve the success of drug discovery campaigns for the treatment of Parkinson’s disease. We measure FRET by fluorescence lifetime, rather than traditional fluorescence intensity, providing a structural readout with greater resolution and precision. This facilitates identification of compounds that cause subtle but significant conformational changes in the ensemble of oligomeric states that are easily missed using intensity-based FRET. We screened a 1280-compound small-molecule library and identified 21 compounds that changed the lifetime by >5 SD. Two of these compounds have nanomolar potency in protecting SH-SY5Y cells from αSN-induced death, providing a nearly tenfold improvement over known inhibitors. We tested the efficacy of several compounds in a primary mouse neuron assay of αSN pathology (phosphorylation of mouse αSN pre-formed fibrils) and show rescue of pathology for two of them. These hits were further characterized with biophysical and biochemical assays to explore potential mechanisms of action. In vitro αSN oligomerization, single-molecule FRET, and protein-observed fluorine NMR experiments demonstrate that these compounds modulate αSN oligomers but not monomers. Subsequent aggregation assays further show that these compounds also deter or block αSN fibril assembly.

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.

scholarly journals Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

2015 ◽  
Vol 112 (38) ◽  
pp. 11835-11840 ◽  
Author(s):  
Yufan He ◽  
Mohammad Mahfuzul Haque ◽  
Dennis J. Stuehr ◽  
H. Peter Lu
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Conformational States ◽  
Calmodulin Binding ◽  
Conformational Fluctuation ◽  
Fluctuation Dynamics ◽  
The Individual ◽  
Domain Motions ◽  
Oxide Synthase

Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS.

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