Disordered proteins follow diverse transition paths as they fold and bind to a partner

Transition paths of macromolecular conformational changes such as protein folding are predicted to be heterogeneous. However, experimental characterization of the diversity of transition paths is extremely challenging because it requires measuring more than one distance during individual transitions. In this work, we used fast three-color single-molecule Förster resonance energy transfer spectroscopy to obtain the distribution of binding transition paths of a disordered protein. About half of the transitions follow a path involving strong non-native electrostatic interactions, resulting in a transition time of 300 to 800 microseconds. The remaining half follow more diverse paths characterized by weaker electrostatic interactions and more than 10 times shorter transition path times. The chain flexibility and non-native interactions make diverse binding pathways possible, allowing disordered proteins to bind faster than folded proteins.

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Ligand-induced transmembrane conformational coupling in monomeric EGFR

10.1101/2021.10.28.466294 ◽

2021 ◽

Author(s):

Shwetha Srinivasan ◽

Raju Regmi ◽

Xingcheng Lin ◽

Courtney A. Dreyer ◽

Xuyan Chen ◽

...

Keyword(s):

Single Molecule ◽

Conformational Changes ◽

Electrostatic Interactions ◽

Transmembrane Helix ◽

Resonance Energy Transfer ◽

Growth Factor Receptor ◽

Resonance Energy ◽

Single Pass ◽

Receptor Interactions ◽

Conformational Coupling

Single pass cell surface receptors regulate cellular processes by transmitting ligand-encoded signals across the plasma membrane via changes to their extracellular and intracellular conformations. While receptor-receptor interactions are established as key aspects of transmembrane signaling, the contribution from the single helix of a monomeric receptor has been challenging to isolate due to the complexity and ligand-dependence of the receptor-receptor interactions. By combining membrane nanodiscs produced with cell-free expression, single-molecule Förster Resonance Energy Transfer measurements, and molecular dynamics simulations, we report that ligand binding induces intracellular conformational changes within monomeric, full-length epidermal growth factor receptor (EGFR). Our observations establish the existence of extracellular/intracellular conformational coupling within a single receptor molecule. We implicate a series of electrostatic interactions in the conformational coupling and find the coupling is inhibited by targeted therapeutics and mutations that also inhibit phosphorylation in cells. Collectively, these results introduce a facile mechanism to link the extracellular and intracellular regions through the single transmembrane helix of monomeric EGFR, and raise the possibility that intramolecular transmembrane conformational changes are common to single-pass membrane proteins.

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

Biological Chemistry ◽

10.1515/bc.2011.001 ◽

2011 ◽

Vol 392 (1-2) ◽

Cited By ~ 13

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.

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Single Molecule FRET: A Powerful Tool to Study Intrinsically Disordered Proteins

Biomolecules ◽

10.3390/biom8040140 ◽

2018 ◽

Vol 8 (4) ◽

pp. 140 ◽

Cited By ~ 17

Author(s):

Sharonda LeBlanc ◽

Prakash Kulkarni ◽

Keith Weninger

Keyword(s):

Single Molecule ◽

Biological Networks ◽

Intrinsically Disordered Proteins ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Polymer Physics ◽

Disordered Proteins ◽

Intrinsically Disordered ◽

Single Molecule Fret ◽

Random Fluctuations

Intrinsically disordered proteins (IDPs) are often modeled using ideas from polymer physics that suggest they smoothly explore all corners of configuration space. Experimental verification of this random, dynamic behavior is difficult as random fluctuations of IDPs cannot be synchronized across an ensemble. Single molecule fluorescence (or Förster) resonance energy transfer (smFRET) is one of the few approaches that are sensitive to transient populations of sub-states within molecular ensembles. In some implementations, smFRET has sufficient time resolution to resolve transitions in IDP behaviors. Here we present experimental issues to consider when applying smFRET to study IDP configuration. We illustrate the power of applying smFRET to IDPs by discussing two cases in the literature of protein systems for which smFRET has successfully reported phosphorylation-induced modification (but not elimination) of the disordered properties that have been connected to impacts on the related biological function. The examples we discuss, PAGE4 and a disordered segment of the GluN2B subunit of the NMDA receptor, illustrate the great potential of smFRET to inform how IDP function can be regulated by controlling the detailed ensemble of disordered states within biological networks.

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

npj Parkinson s Disease ◽

10.1038/s41531-021-00195-6 ◽

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.

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Integrating multiple experimental data to determine conformational ensembles of an intrinsically disordered protein

10.1101/2020.02.05.935890 ◽

2020 ◽

Cited By ~ 2

Author(s):

Gregory-Neal W. Gomes ◽

Mickaël Krzeminski ◽

Ashley Namini ◽

Erik. W. Martin ◽

Tanja Mittag ◽

...

Keyword(s):

Experimental Data ◽

Single Molecule ◽

Intrinsically Disordered Proteins ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Intrinsically Disordered Protein ◽

Disordered Proteins ◽

Saxs Data ◽

Conformational Ensembles ◽

Intrinsically Disordered

AbstractIntrinsically disordered proteins (IDPs) have fluctuating heterogeneous conformations, which makes structural characterization challenging, but of great interest, since their conformational ensembles are the link between their sequences and functions. An accurate description of IDP conformational ensembles depends crucially on the amount and quality of the experimental data, how it is integrated, and if it supports a consistent structural picture. We have used an integrative modelling approach to understand how conformational restraints imposed by the most common structural techniques for IDPs: Nuclear Magnetic Resonance (NMR) spectroscopy, Small-angle X-ray Scattering (SAXS), and single-molecule Förster Resonance Energy Transfer (smFRET) reach concordance on structural ensembles for Sic1 and phosphorylated Sic1 (pSic1). To resolve apparent discrepancies between smFRET and SAXS, we integrated SAXS data with non-smFRET (NMR) data and reserved the new smFRET data for Sic1 and pSic1 as an independent validation. The consistency of the SAXS/NMR restrained ensembles with smFRET, which was not guaranteed a priori, indicates that the perturbative effects of NMR or smFRET labels on the Sic1 and pSic1 ensembles are minimal. Furthermore, the mutual agreement with such a diverse set of experimental data suggest that details of the generated ensembles can now be examined with a high degree of confidence to reveal distinguishing features of Sic1 vs. pSic1. From the experimentally well supported ensembles, we find they are consistent with independent biophysical models of Sic1’s ultrasensitive binding to its partner Cdc4. Our results underscore the importance of integrative modelling in calculating and drawing biological conclusions from IDP conformational ensembles.

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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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