scholarly journals Probing the Structural Dynamics of the Activation Gate of KcsA Using Homo-FRET Measurements

2021 ◽  
Vol 22 (21) ◽  
pp. 11954
Author(s):  
Clara Díaz-García ◽  
Maria Lourdes Renart ◽  
José Antonio Poveda ◽  
Ana Marcela Giudici ◽  
José M. González-Ros ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Allosteric Coupling ◽  
Allosteric Communication ◽  
Activation Gate ◽  
Fret Efficiency ◽  
High Degree

The allosteric coupling between activation and inactivation processes is a common feature observed in K+ channels. Particularly, in the prokaryotic KcsA channel the K+ conduction process is controlled by the inner gate, which is activated by acidic pH, and by the selectivity filter (SF) or outer gate, which can adopt non-conductive or conductive states. In a previous study, a single tryptophan mutant channel (W67 KcsA) enabled us to investigate the SF dynamics using time-resolved homo-Förster Resonance Energy Transfer (homo-FRET) measurements. Here, the conformational changes of both gates were simultaneously monitored after labelling the G116C position with tetramethylrhodamine (TMR) within a W67 KcsA background. At a high degree of protein labeling, fluorescence anisotropy measurements showed that the pH-induced KcsA gating elicited a variation in the homo-FRET efficiency among the conjugated TMR dyes (TMR homo-FRET), while the conformation of the SF was simultaneously tracked (W67 homo-FRET). The dependence of the activation pKa of the inner gate with the ion occupancy of the SF unequivocally confirmed the allosteric communication between the two gates of KcsA. This simple TMR homo-FRET based ratiometric assay can be easily extended to study the conformational dynamics associated with the gating of other ion channels and their modulation.

scholarly journals Conformational dynamics of Cas9 governing DNA cleavage revealed by single molecule FRET

2017 ◽  
Author(s):  
Mengyi Yang ◽  
Sijia Peng ◽  
Ruirui Sun ◽  
Jingdi Lin ◽  
Nan Wang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Cleavage ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Nuclease Activity ◽  
Single Molecule Fluorescence ◽  
Single Molecule Fret ◽  
Allosteric Communication ◽  
Target Binding

SummaryOff-target binding and cleavage by Cas9 pose as major challenges in its applications. How conformational dynamics of Cas9 governs its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms all spontaneously transits between three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We furthermore uncovered a surprising long-range allosteric communication between the HNH domain and RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox.

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.

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 In Vitro Assays for the Discovery of PCSK9 Autoprocessing Inhibitors

2016 ◽  
Vol 21 (10) ◽  
pp. 1034-1041 ◽  
Author(s):  
Scott P. Salowe ◽  
Lei Zhang ◽  
Hratch J. Zokian ◽  
Jennifer J. Gesell ◽  
Deborah L. Zink ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Resonance Energy Transfer ◽  
Density Lipoprotein ◽  
Resonance Energy ◽  
In Vitro Study ◽  
In Vitro Assays ◽  
Time Resolved

PCSK9 plays a significant role in regulating low-density lipoprotein (LDL) cholesterol levels and has become an important drug target for treating hypercholesterolemia. Although a member of the serine protease family, PCSK9 only catalyzes a single reaction, the autocleavage of its prodomain. The maturation of the proprotein is an essential prerequisite for the secretion of PCSK9 to the extracellular space where it binds the LDL receptor and targets it for degradation. We have found that a construct of proPCSK9 where the C-terminal domain has been truncated has sufficient stability to be expressed and purified from Escherichia coli for the in vitro study of autoprocessing. Using automated Western analysis, we demonstrate that autoprocessing exhibits the anticipated first-order kinetics. A high-throughput time-resolved fluorescence resonance energy transfer assay for autocleavage has been developed using a PCSK9 monoclonal antibody that is sensitive to the conformational changes that occur upon maturation of the proprotein. Kinetic theory has been developed that describes the behavior of both reversible and irreversible inhibitors of autocleavage. The analysis of an irreversible lactone inhibitor validates the expected relationship between potency and the reaction end point. An orthogonal liquid chromatography–mass spectrometry assay has also been implemented for the confirmation of hits from the antibody-based assays.

scholarly journals Real-time Conformational Dynamics of SARS-CoV-2 Spikes on Virus Particles

2020 ◽  
Author(s):  
Maolin Lu ◽  
Pradeep D. Uchil ◽  
Wenwei Li ◽  
Desheng Zheng ◽  
Daniel S. Terry ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Conformational Changes ◽  
Vaccine Development ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Virus Particles ◽  
Immunogen Design ◽  
Real Time Information

AbstractSARS-CoV-2 spike (S) mediates entry into cells and is critical for vaccine development against COVID-19. Structural studies have revealed distinct conformations of S, but real-time information that connects these structures, is lacking. Here we apply single-molecule Förster Resonance Energy Transfer (smFRET) imaging to observe conformational dynamics of S on virus particles. Virus-associated S dynamically samples at least four distinct conformational states. In response to hACE2, S opens sequentially into the hACE2-bound S conformation through at least one on-path intermediate. Conformational preferences of convalescent plasma and antibodies suggest mechanisms of neutralization involving either competition with hACE2 for binding to RBD or allosteric interference with conformational changes required for entry. Our findings inform on mechanisms of S recognition and conformations for immunogen design.

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

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 56
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.

scholarly journals Conformational changes in Arp2/3 complex induced by ATP, WASp-VCA and actin filaments

2017 ◽  
Author(s):  
Sofia Espinoza-Sanchez ◽  
Lauren Ann Metskas ◽  
Steven Z. Chou ◽  
Elizabeth Rhoades ◽  
Thomas D. Pollard
Keyword(s):  
Electron Microscopy ◽  
Energy Transfer ◽  
Structural Change ◽  
Fluorescence Spectroscopy ◽  
Conformational Changes ◽  
Actin Filaments ◽  
Fluorescent Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fret Efficiency

AbstractWe used fluorescence spectroscopy and electron microscopy to determine how binding of ATP, nucleation-promoting factors (NPF), actin monomers and actin filaments change the conformation of Arp2/3 complex during the process that nucleates an actin filament branch. We mutated subunits of Schizosaccharomyces pombe Arp2/3 complex for labeling with fluorescent dyes at either the C-termini of Arp2 and Arp3 or ArpC1 and ArpC3. We measured Förster resonance energy transfer (FRET) efficiency (ETeff) between the dyes in the presence of the various ligands. We also computed class averages from electron micrographs of negatively stained specimens. ATP binding made small conformational changes of the nucleotide binding clefts of the Arp subunits. WASp-VCA, WASp-CA, and WASp-actin-VCA changed the ETeff between the dyes on the Arp2 and Arp3 subunits much more than between dyes on ArpC1 and ArpC3. Ensemble FRET detected a different structural change that involves bringing ArpC1 and ArpC3 closer together when Arp2/3 complex bound actin filaments. Each of the ligands that activates Arp2/3 complex changes the structure in different ways, each leading progressively to fully activated Arp2/3 complex on the side of a filament.

scholarly journals DNA polymerase β fingers movement revealed by single-molecule FRET suggests a partially closed conformation as a fidelity checkpoint

2018 ◽  
Author(s):  
Carel Fijen ◽  
Mariam Mahmoud ◽  
Rebecca Kaup ◽  
Jamie Towle-Weicksel ◽  
Joann Sweasy ◽  
...  
Keyword(s):  
Dna Repair ◽  
Dna Polymerase ◽  
Single Molecule ◽  
Conformational Changes ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dna Polymerase Β ◽  
Gapped Dna ◽  
Dna Substrate

The eukaryotic DNA polymerase β plays an important role in cellular DNA repair as it fills gaps in single nucleotide gapped DNA that result from removal of damaged bases. Since defects in DNA repair may lead to cancer and genetic instabilities, Pol β has been extensively studied, especially substrate binding and a fidelity-related conformational change called fingers closing. Here, we applied single-molecule Förster resonance energy transfer to study the conformational dynamics of Pol β. Using an acceptor labelled polymerase and a donor labelled DNA substrate, we measured distance changes associated with DNA binding and fingers movement. Our findings suggest that Pol β does not bend its gapped DNA substrate to the extent related crystal structures indicate: instead, bending seems to be significantly less profound. Furthermore, we visualized dynamic fingers closing in single Pol β-DNA complexes upon addition of complementary nucleotides and derived rates of conformational changes. Additionally, we provide evidence that the fingers close only partially when an incorrect nucleotide is bound. This ajar conformation found in Pol β, a polymerase of the X-family, suggests the existence of an additional fidelity checkpoint similar to what has been previously proposed for a member of the A-family, the bacterial DNA polymerase I.

scholarly journals Real-Time Analysis of Individual Ebola Virus Glycoproteins Reveals Pre-Fusion, Entry-Relevant Conformational Dynamics

Viruses ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 103 ◽  
Author(s):  
Natasha D. Durham ◽  
Angela R. Howard ◽  
Ramesh Govindan ◽  
Fernando Senjobe ◽  
J. Maximilian Fels ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Neutralizing Antibodies ◽  
Ebola Virus ◽  
Transmembrane Protein ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Target Cells ◽  
Viral Fusion

The Ebola virus (EBOV) envelope glycoprotein (GP) mediates the fusion of the virion membrane with the membrane of susceptible target cells during infection. While proteolytic cleavage of GP by endosomal cathepsins and binding of the cellular receptor Niemann-Pick C1 protein (NPC1) are essential steps for virus entry, the detailed mechanisms by which these events promote membrane fusion remain unknown. Here, we applied single-molecule Förster resonance energy transfer (smFRET) imaging to investigate the structural dynamics of the EBOV GP trimeric ectodomain, and the functional transmembrane protein on the surface of pseudovirions. We show that in both contexts, pre-fusion GP is dynamic and samples multiple conformations. Removal of the glycan cap and NPC1 binding shift the conformational equilibrium, suggesting stabilization of conformations relevant to viral fusion. Furthermore, several neutralizing antibodies enrich alternative conformational states. This suggests that these antibodies neutralize EBOV by restricting access to GP conformations relevant to fusion. This work demonstrates previously unobserved dynamics of pre-fusion EBOV GP and presents a platform with heightened sensitivity to conformational changes for the study of GP function and antibody-mediated neutralization.

scholarly journals Kinetics of the conformational cycle of Hsp70 reveals the importance of the dynamic and heterogeneous nature of Hsp70 for its function

2020 ◽  
Vol 117 (14) ◽  
pp. 7814-7823 ◽  
Author(s):  
Si Wu ◽  
Liu Hong ◽  
Yuqing Wang ◽  
Jieqiong Yu ◽  
Jie Yang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Substrate Binding ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bound State ◽  
Kinetic Measurements ◽  
Kinetics Of ◽  
Conformational Distribution

Hsp70 is a conserved molecular chaperone that plays an indispensable role in regulating protein folding, translocation, and degradation. The conformational dynamics of Hsp70 and its regulation by cochaperones are vital to its function. Using bulk and single-molecule fluorescence resonance energy transfer (smFRET) techniques, we studied the interdomain conformational distribution of human stress-inducible Hsp70A1 and the kinetics of conformational changes induced by nucleotide and the Hsp40 cochaperone Hdj1. We found that the conformations between and within the nucleotide- and substrate-binding domains show heterogeneity. The conformational distribution in the ATP-bound state can be induced by Hdj1 to form an “ADP-like” undocked conformation, which is an ATPase-stimulated state. Kinetic measurements indicate that Hdj1 binds to monomeric Hsp70 as the first step, then induces undocking of the two domains and closing of the substrate-binding cleft. Dimeric Hdj1 then facilitates dimerization of Hsp70 and formation of a heterotetrameric Hsp70–Hsp40 complex. Our results provide a kinetic view of the conformational cycle of Hsp70 and reveal the importance of the dynamic nature of Hsp70 for its function.

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