scholarly journals Single-molecule transport kinetics of a glutamate transporter homolog shows static disorder

2020 ◽  
Vol 6 (22) ◽  
pp. eaaz1949 ◽  
Author(s):  
Didar Ciftci ◽  
Gerard H. M. Huysmans ◽  
Xiaoyu Wang ◽  
Changhao He ◽  
Daniel Terry ◽  
...  
Keyword(s):  
Single Molecule ◽  
Single Channel ◽  
Conformational Dynamics ◽  
Glutamate Transporter ◽  
Resonance Energy ◽  
Fold Increase ◽  
Transport Kinetics ◽  
Kinetic Properties ◽  
Molecule Transport ◽  
Kinetics Of

Kinetic properties of membrane transporters are typically poorly defined because high-resolution functional assays analogous to single-channel recordings are lacking. Here, we measure single-molecule transport kinetics of a glutamate transporter homolog from Pyrococcus horikoshii, GltPh, using fluorescently labeled periplasmic amino acid binding protein as a fluorescence resonance energy transfer–based sensor. We show that individual transporters can function at rates varying by at least two orders of magnitude that persist for multiple turnovers. A gain-of-function mutant shows increased population of the fast-acting transporters, leading to a 10-fold increase in the mean transport rate. These findings, which are broadly consistent with earlier single-molecule measurements of GltPh conformational dynamics, suggest that GltPh transport is defined by kinetically distinct populations that exhibit long-lasting “molecular memory.”

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.

Monitoring the Structural Dynamics of U2 snRNA Via Single-Molecule Förster Resonance Energy Transfer

2018 ◽  
Author(s):  
Alexander Carl DeHaven
Keyword(s):  
Energy Transfer ◽  
Structural Dynamics ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Förster Resonance Energy Transfer ◽  
U2 Snrna ◽  
Folding Dynamics ◽  
The Impact

This thesis contains four topic areas: a review of single-molecule microscropy methods and splicing, conformational dynamics of stem II of the U2 snRNA, the impact of post-transcriptional modifications on U2 snRNA folding dynamics, and preliminary findings on Mango aptamer folding dynamics.

scholarly journals Allosteric modulation of the SARS-CoV-2 spike conformation

2021 ◽  
Author(s):  
Marco A Diaz-Salinas ◽  
Qi Li ◽  
Monir Ejemel ◽  
Yang Wang ◽  
James B Munro
Keyword(s):  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Structural Data ◽  
Correlation Spectroscopy ◽  
Host Cells ◽  
Parallel Solution ◽  
Real Time Analysis ◽  
Multiple Conformations

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells through binding to angiotensin-converting enzyme 2 (ACE2), which is mediated by the receptor-binding domain (RBD) of the viral spike (S) glycoprotein. Structural data and real-time analysis of conformational dynamics have shown that S can adopt multiple conformations, which mediate the exposure of the ACE2-binding site in the RBD. Here, using single-molecule Förster resonance energy transfer (smFRET) imaging we report the effects of ACE2 and antibody binding on the conformational dynamics of S from the Wuhan-1 strain and the B.1 variant (D614G). We found that antibodies that target diverse epitopes, including those distal to the RBD, stabilize the RBD in a position competent for ACE2 binding. Parallel solution-based binding experiments using fluorescence correlation spectroscopy (FCS) indicated antibody-mediated enhancement of ACE2 binding. These findings inform on novel strategies for therapeutic antibody cocktails.

scholarly journals Accurate Determination of Human CPR Conformational Equilibrium by smFRET using Dual Orthogonal Non-Canonical Amino Acid Labeling

2018 ◽  
Author(s):  
Robert B. Quast ◽  
Fataneh Fatemi ◽  
Michel Kranendonk ◽  
Emmanuel Margeat ◽  
Gilles Truan
Keyword(s):  
Single Molecule ◽  
Conformational Equilibrium ◽  
Fluorescent Dyes ◽  
Photophysical Properties ◽  
Accurate Determination ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
General Strategy

ABSTRACTConjugation of fluorescent dyes to proteins - a prerequisite for the study of conformational dynamics by single molecule Förster resonance energy transfer (smFRET) - can lead to substantial changes of the dye’s photophysical properties, ultimately biasing the quantitative determination of inter-dye distances. In particular the popular cyanine dyes and their derivatives, which are by far the most used dyes in smFRET experiments, exhibit such behavior. To overcome this, a general strategy to site-specifically equip proteins with FRET pairs by chemo-selective reactions using two distinct non-canonical amino acids simultaneously incorporated through genetic code expansion in Escherichia coli was developed. Applied to human NADPH- cytochrome P450 reductase (CPR), the importance of homogenously labeled samples for accurate determination of FRET efficiencies was demonstrated. Furthermore, the effect of NADP+ on the ionic strength dependent modulation of the conformational equilibrium of CPR was unveiled. Given its generality and accuracy, the presented methodology establishes a new benchmark to decipher complex molecular dynamics on single molecules.

scholarly journals The mechanochemistry of the kinesin-2 KIF3AC heterodimer is related to strain-dependent kinetic properties of KIF3A and KIF3C

2020 ◽  
Vol 117 (27) ◽  
pp. 15632-15641
Author(s):  
Brandon M. Bensel ◽  
Michael S. Woody ◽  
Serapion Pyrpassopoulos ◽  
Yale E. Goldman ◽  
Susan P. Gilbert ◽  
...  
Keyword(s):  
Single Molecule ◽  
Mechanical Load ◽  
Optical Trap ◽  
Kinetic Properties ◽  
Dependent Manner ◽  
Force Sensitivity ◽  
Mammalian Neuron ◽  
Kinetics Of ◽  
Strain Dependent ◽  
Transport Potential

KIF3AC is a mammalian neuron-specific kinesin-2 implicated in intracellular cargo transport. It is a heterodimer of KIF3A and KIF3C motor polypeptides which have distinct biochemical and motile properties as engineered homodimers. Single-molecule motility assays show that KIF3AC moves processively along microtubules at a rate faster than expected given the motility rates of the KIF3AA and much slower KIF3CC homodimers. To resolve the stepping kinetics of KIF3A and KIF3C motors in homo- and heterodimeric constructs and determine their transport potential under load, we assayed motor activity using interferometric scattering microscopy and optical trapping. The distribution of stepping durations of KIF3AC molecules is described by a rate (k1= 11 s−1) without apparent kinetic asymmetry. Asymmetry was also not apparent under hindering or assisting mechanical loads in the optical trap. KIF3AC shows increased force sensitivity relative to KIF3AA yet is more capable of stepping against mechanical load than KIF3CC. Interestingly, the behavior of KIF3C mirrors prior studies of kinesins with increased interhead compliance. Microtubule gliding assays containing 1:1 mixtures of KIF3AA and KIF3CC result in speeds similar to KIF3AC, suggesting the homodimers mechanically impact each other’s motility to reproduce the behavior of the heterodimer. Our observations are consistent with a mechanism in which the stepping of KIF3C can be activated by KIF3A in a strain-dependent manner, similar to application of an assisting load. These results suggest that the mechanochemical properties of KIF3AC can be explained by the strain-dependent kinetics of KIF3A and KIF3C.

scholarly journals Labeling of Proteins for Single-Molecule Fluorescence Spectroscopy

2020 ◽  
Author(s):  
Franziska Zosel ◽  
Andrea Holla ◽  
Benjamin Schuler
Keyword(s):  
Fluorescence Spectroscopy ◽  
Single Molecule ◽  
Intrinsically Disordered Proteins ◽  
Conformational Dynamics ◽  
Control Sample ◽  
Resonance Energy ◽  
Disordered Proteins ◽  
Single Molecule Fluorescence ◽  
Intrinsically Disordered ◽  
Single Molecule Fluorescence Spectroscopy

Single-molecule fluorescence spectroscopy has become an important technique for studying the conformational dynamics and folding of proteins. A key step for performing such experiments is the availability of high-quality samples. Here we describe the practical details of a simple and widely applicable strategy for preparing proteins that are site-specifically labeled with a donor and an acceptor dye for single-molecule Förster resonance energy transfer (FRET) experiments. The method is based on introducing two cysteine residues that are labeled with maleimide-functionalized fluorophores, combined with high-resolution chromatography. We discuss how to optimize site-specific labeling even in the absence of orthogonal coupling chemistry and present purification strategies that are suitable for samples ranging from intrinsically disordered proteins to large folded proteins. We also discuss common problems in protein labeling, how to avoid them, and how to stringently control sample quality.<br>

scholarly journals Conformational dynamics of auto-inhibition in the ER calcium sensor STIM1

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Stijn van Dorp ◽  
Ruoyi Qiu ◽  
Ucheor B Choi ◽  
Minnie M Wu ◽  
Michelle Yen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bound State ◽  
Structural Basis ◽  
Resting Cells ◽  
Dual Roles ◽  
Er Calcium

The dimeric ER Ca2+ sensor STIM1 controls store-operated Ca2+ entry (SOCE) through the regulated binding of its CRAC activation domain (CAD) to Orai channels in the plasma membrane. In resting cells, the STIM1 CC1 domain interacts with CAD to suppress SOCE, but the structural basis of this interaction is unclear. Using single-molecule Förster resonance energy transfer (smFRET) and protein crosslinking approaches, we show that CC1 interacts dynamically with CAD in a domain-swapped configuration with an orientation predicted to sequester its Orai-binding region adjacent to the ER membrane. Following ER Ca2+ depletion and release from CAD, cysteine crosslinking indicates that the two CC1 domains become closely paired along their entire length in the active Orai-bound state. These findings provide a structural basis for the dual roles of CC1: sequestering CAD to suppress SOCE in resting cells and propelling it towards the plasma membrane to activate Orai and SOCE after store depletion.

scholarly journals A Splice Site-Sensing Conformational Switch in U2AF2 is Modulated by U2AF1 and its Recurrent Myelodysplasia-Associated Mutation

2019 ◽  
Author(s):  
Chandani Warnasooriya ◽  
Eliezra Glasser ◽  
Callen F. Feeney ◽  
Kholiswa M. Laird ◽  
Jermaine L. Jenkins ◽  
...  
Keyword(s):  
Single Molecule ◽  
Splice Site ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Splicing Factors ◽  
Rna Recognition ◽  
Rna Recognition Motifs ◽  
Recognition Motifs ◽  
Rna Complexes

ABSTRACTAn essential heterodimer of the U2AF1 and U2AF2 pre-mRNA splicing factors nucleates spliceosome assembly at polypyrimidine (Py) signals preceding the major class of 3ʹ splice sites. Among myelodysplastic syndromes (MDS), U2AF1 frequently acquires an S34F-encoding mutation. The influence of the U2AF1 subunit and its S34F mutation on the U2AF2 conformations remains unknown. Here, we employ single molecule Förster resonance energy transfer (FRET) to determine the influence of wild-type or S34F-substituted U2AF1 on the conformational dynamics of U2AF2 and its splice site RNA complexes. In the absence of RNA, the U2AF1 subunit stabilizes a high FRET value, which by structure-guided mutagenesis corresponds to a closed conformation of the tandem U2AF2 RNA recognition motifs (RRMs). When the U2AF heterodimer is bound to a strong, uridine-rich splice site, U2AF2 switches to a lower FRET value characteristic of an open, side-by-side arrangement of the RRMs. Remarkably, the U2AF heterodimer binds weak, uridine-poor Py tracts as a mixture of closed and open U2AF2 conformations, which are modulated by the S34F mutation. Shifts between open and closed U2AF2 may underlie U2AF1-dependent splicing of degenerate Py tracts and contribute to a subset of S34F-dysregulated splicing events in MDS patients.

scholarly journals Strain-Dependent Kinetic Properties of KIF3A and KIF3C Tune the Mechanochemistry of the KIF3AC Heterodimer

2019 ◽  
Author(s):  
Brandon M. Bensel ◽  
Michael S. Woody ◽  
Serapion Pyrpassopoulos ◽  
Yale E. Goldman ◽  
Susan P. Gilbert ◽  
...  
Keyword(s):  
Single Molecule ◽  
Mechanical Load ◽  
Optical Trap ◽  
Slow Step ◽  
Kinetic Properties ◽  
Dependent Manner ◽  
Cargo Transport ◽  
Mammalian Neuron ◽  
Kinetics Of ◽  
Strain Dependent

AbstractKIF3AC is a mammalian neuron-specific kinesin-2 implicated in intracellular cargo transport. It is a heterodimer of KIF3A and KIF3C motor polypeptides which have distinct biochemical and motile properties as engineered homodimers. Single-molecule motility assays show that KIF3AC moves processively along microtubules at a rate faster than expected given the motility rates of the KIF3AA and much slower KIF3CC homodimers. To resolve the stepping kinetics of KIF3A and KIF3C motors in homo-and heterodimeric constructs, and to determine their transport potential under mechanical load, we assayed motor activity using interferometric scattering (iSCAT) microscopy and optical trapping. The distribution of stepping durations of KIF3AC molecules is described by a rate (k1 = 11 s−1) without apparent kinetic asymmetry in stepping. Asymmetry was also not apparent under hindering or assisting mechanical loads of 1 pN in the optical trap. KIF3AC shows increased force sensitivity relative to KIF3AA, yet is more capable of stepping against mechanical load than KIF3CC. Microtubule gliding assays containing 1:1 mixtures of KIF3AA and KIF3CC result in speeds similar to KIF3AC, indicating the homodimers mechanically impact each other’s motility to reproduce the behavior of the heterodimer. We conclude that the stepping of KIF3C can be activated by KIF3A in a strain-dependent manner which is similar to application of an assisting load, and the behavior of KIF3C mirrors prior studies of kinesins with increased interhead compliance. These results suggest that KIF3AC-based cargo transport likely requires multiple motors, and its mechanochemical properties arise due to the strain-dependences of KIF3A and KIF3C.Significance StatementKinesins are important long-range intracellular transporters in neurons required by the extended length of the axon and dendrites and selective cargo transport to each. The mammalian kinesin-2, KIF3AC, is a neuronal heterodimer of fast and slow motor polypeptides. Our results show that KIF3AC has a single observed stepping rate in the presence and absence of load and detaches from the microtubule rapidly under load. Interestingly, both KIF3A and assisting loads accelerate the kinetics of KIF3C. These results suggest that KIF3AC is an unconventional cargo transporter and its motile properties do not represent a combination of alternating fast and slow step kinetics. We demonstrate that the motile properties of KIF3AC represent a mechanochemistry that is specific to KIF3AC and may provide functional advantages in neurons.

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