scholarly journals The allosteric mechanism of substrate-specific transport in SLC6 is mediated by a volumetric sensor

2019 ◽  
Vol 116 (32) ◽  
pp. 15947-15956 ◽  
Author(s):  
Michael V. LeVine ◽  
Daniel S. Terry ◽  
George Khelashvili ◽  
Zarek S. Siegel ◽  
Matthias Quick ◽  
...  
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Binding Pocket ◽  
Coupled Transport ◽  
Specific Transport ◽  
Slc6 Family ◽  
Transport Of Ions ◽  
The Stability ◽  
Dynamics Simulations

Neurotransmitter:sodium symporters (NSSs) in the SLC6 family terminate neurotransmission by coupling the thermodynamically favorable transport of ions to the thermodynamically unfavorable transport of neurotransmitter back into presynaptic neurons. Results from many structural, functional, and computational studies on LeuT, a bacterial NSS homolog, have provided critical insight into the mechanism of sodium-coupled transport, but the mechanism underlying substrate-specific transport rates is still not understood. We present a combination of molecular dynamics simulations, single-molecule fluorescence resonance energy transfer (smFRET) imaging, and measurements of Na+ binding and substrate transport that reveals an allosteric substrate specificity mechanism. In this mechanism, residues F259 and I359 in the substrate binding pocket couple the binding of substrate to Na+ release from the Na2 site by allosterically modulating the stability of a partially open, inward-facing state. We propose a model for transport selectivity in which residues F259 and I359 act as a volumetric sensor that inhibits the transport of bulky amino acids.

scholarly journals The allosteric mechanism of substrate-specific transport in SLC6 is mediated by a volumetric sensor

2019 ◽  
Author(s):  
Michael V. LeVine ◽  
Daniel S. Terry ◽  
George Khelashvili ◽  
Zarek S. Siegel ◽  
Matthias Quick ◽  
...  
Keyword(s):  
Single Molecule ◽  
Substrate Binding ◽  
Md Simulations ◽  
Binding Pocket ◽  
Coupled Transport ◽  
Single Molecule Fret ◽  
Allosteric Mechanism ◽  
Specific Transport ◽  
Slc6 Family ◽  
The Stability

AbstractNeurotransmitter:sodium symporters (NSS) in the SLC6 family terminate neurotransmission by coupling the thermodynamically favorable transport of ions to the thermodynamically unfavorable transport of neurotransmitter back into presynaptic neurons. While a combination of structural, functional, and computational studies on LeuT, a bacterial NSS homolog, has provided critical insight into the mechanism of sodium-coupled transport, the mechanism underlying substrate-specific transport rates is still not understood. We present a combination of MD simulations, single-molecule FRET imaging, and measurements of Na+ binding and substrate transport that reveal an allosteric mechanism in which residues F259 and I359 in the substrate binding pocket couple substrate binding to Na+ release from the Na2 site through allosteric modulation of the stability of a partially-open, inward-facing state. We propose a new model for transport selectivity in which the two residues act as a volumetric sensor that inhibits the transport of bulky amino acids.

Structural dynamics of P-type ATPase ion pumps

2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
New Technologies ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Dynamics Simulations ◽  
Types Of Information ◽  
P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

scholarly journals Rational design of protein-specific folding modifiers

2020 ◽  
Author(s):  
Anirban Das ◽  
Anju Yadav ◽  
Mona Gupta ◽  
R Purushotham ◽  
Vishram L. Terse ◽  
...  
Keyword(s):  
Single Molecule ◽  
Rational Design ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Force Measurements ◽  
Folding Nucleus ◽  
Dynamics Simulations ◽  
General Method

AbstractProtein folding can go wrong in vivo and in vitro, with significant consequences for the living cell and the pharmaceutical industry, respectively. Here we propose a general design principle for constructing small peptide-based protein-specific folding modifiers. We construct a ‘xenonucleus’, which is a pre-folded peptide that resembles the folding nucleus of a protein, and demonstrate its activity on the folding of ubiquitin. Using stopped-flow kinetics, NMR spectroscopy, Förster Resonance Energy transfer, single-molecule force measurements, and molecular dynamics simulations, we show that the ubiquitin xenonucleus can act as an effective decoy for the native folding nucleus. It can make the refolding faster by 33 ± 5% at 3 M GdnHCl. In principle, our approach provides a general method for constructing specific, genetically encodable, folding modifiers for any protein which has a well-defined contiguous folding nucleus.

scholarly journals Cohesin-dockerin code in cellulosomal dual binding modes and its allosteric regulation by proline isomerization

2019 ◽  
Author(s):  
Andrés Manuel Vera ◽  
Albert Galera-Prat ◽  
Michał Wojciechowski ◽  
Bartosz Różycki ◽  
Douglas Vinson Laurents ◽  
...  
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Binding Modes ◽  
Prolyl Isomerase ◽  
Proline Isomerization ◽  
Allosteric Control ◽  
Dynamics Simulations ◽  
Enzyme Complexes

AbstractCellulose is the most abundant organic molecule on Earth and represents a renewable and practically everlasting feedstock for the production of biofuels and chemicals. Self-assembled owing to the high-affinity cohesin-dockerin interaction, cellulosomes are huge multi-enzyme complexes with unmatched efficiency in the degradation of recalcitrant lignocellulosic substrates. The recruitment of diverse dockerin-borne enzymes into a multicohesin protein scaffold dictates the three-dimensional layout of the complex, and interestingly two alternative binding modes have been proposed. Using single-molecule Fluorescence Resonance Energy Transfer, molecular dynamics simulations and NMR measurements on a range of cohesin-dockerin pairs, we directly detect varying distributions between these binding modes that follow a built-in cohesin-dockerin code. Surprisingly, we uncover a prolyl isomerase-modulated allosteric control mechanism, mediated by the isomerization state of a single proline residue, which regulates the distribution and kinetics of binding modes. Overall, our data provide a novel mechanistic understanding of the structural plasticity and dynamics of cellulosomes.

scholarly journals Molecular mechanism of substrate recognition and transport by the AtSWEET13 sugar transporter

2017 ◽  
Vol 114 (38) ◽  
pp. 10089-10094 ◽  
Author(s):  
Lei Han ◽  
Yongping Zhu ◽  
Min Liu ◽  
Ye Zhou ◽  
Guangyuan Lu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Binding Pocket ◽  
Phloem Loading ◽  
Order Structure ◽  
Substrate Binding Pocket ◽  
Sugar Transporters ◽  
Substrate Analog ◽  
Pollen Nutrition

Sugar Will Eventually be Exported Transporters (SWEETs) are recently identified sugar transporters that can discriminate and transport di- or monosaccharides across a membrane following the concentration gradient. SWEETs play key roles in plant biological processes, such as pollen nutrition, nectar secretion, seed filling, and phloem loading. SWEET13 fromArabidopsis thaliana(AtSWEET13) is an important sucrose transporter in pollen development. Here, we report the 2.8-Å resolution crystal structure of AtSWEET13 in the inward-facing conformation with a substrate analog, 2′-deoxycytidine 5′-monophosphate, bound in the central cavity. In addition, based on the results of an in-cell transport activity assay and single-molecule Förster resonance energy transfer analysis, we suggest a mechanism for substrate selectivity based on the size of the substrate-binding pocket. Furthermore, AtSWEET13 appears to form a higher order structure presumably related to its function.

scholarly journals Conformational spread and dynamics in allostery of NMDA receptors

2020 ◽  
Vol 117 (7) ◽  
pp. 3839-3847 ◽  
Author(s):  
Ryan J. Durham ◽  
Nabina Paudyal ◽  
Elisa Carrillo ◽  
Nidhi Kaur Bhatia ◽  
David M. Maclean ◽  
...  
Keyword(s):  
Single Molecule ◽  
Transmembrane Domain ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fine Tuning ◽  
Negative Cooperativity ◽  
Dimer Interface ◽  
Transmembrane Segments ◽  
Tightly Coupled ◽  
Dynamics Simulations

Allostery can be manifested as a combination of repression and activation in multidomain proteins allowing for fine tuning of regulatory mechanisms. Here we have used single molecule fluorescence resonance energy transfer (smFRET) and molecular dynamics simulations to study the mechanism of allostery underlying negative cooperativity between the two agonists glutamate and glycine in the NMDA receptor. These data show that binding of one agonist leads to conformational flexibility and an increase in conformational spread at the second agonist site. Mutational and cross-linking studies show that the dimer–dimer interface at the agonist-binding domain mediates the allostery underlying the negative cooperativity. smFRET on the transmembrane segments shows that they are tightly coupled in the unliganded and single agonist-bound form and only upon binding both agonists the transmembrane domain explores looser packing which would facilitate activation.

scholarly journals Quantification and demonstration of the collective constriction-by-ratchet mechanism in the dynamin molecular motor

2021 ◽  
Vol 118 (28) ◽  
pp. e2101144118
Author(s):  
Oleg M. Ganichkin ◽  
Renee Vancraenenbroeck ◽  
Gabriel Rosenblum ◽  
Hagen Hofmann ◽  
Alexander S. Mikhailov ◽  
...  
Keyword(s):  
Single Molecule ◽  
Computational Study ◽  
Molecular Motor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Power Stroke ◽  
Ratchet Mechanism ◽  
Cross Bridges ◽  
Dynamics Simulations ◽  
Gtpase Cycle

Dynamin oligomerizes into helical filaments on tubular membrane templates and, through constriction, cleaves them in a GTPase-driven way. Structural observations of GTP-dependent cross-bridges between neighboring filament turns have led to the suggestion that dynamin operates as a molecular ratchet motor. However, the proof of such mechanism remains absent. Particularly, it is not known whether a powerful enough stroke is produced and how the motor modules would cooperate in the constriction process. Here, we characterized the dynamin motor modules by single-molecule Förster resonance energy transfer (smFRET) and found strong nucleotide-dependent conformational preferences. Integrating smFRET with molecular dynamics simulations allowed us to estimate the forces generated in a power stroke. Subsequently, the quantitative force data and the measured kinetics of the GTPase cycle were incorporated into a model including both a dynamin filament, with explicit motor cross-bridges, and a realistic deformable membrane template. In our simulations, collective constriction of the membrane by dynamin motor modules, based on the ratchet mechanism, is directly reproduced and analyzed. Functional parallels between the dynamin system and actomyosin in the muscle are seen. Through concerted action of the motors, tight membrane constriction to the hemifission radius can be reached. Our experimental and computational study provides an example of how collective motor action in megadalton molecular assemblies can be approached and explicitly resolved.

scholarly journals DNA Origami Voltage Sensors for Transmembrane Potentials with Single-Molecule Sensitivity

2021 ◽  
Author(s):  
Sarah E. Ochmann ◽  
Himanshu Joshi ◽  
Ece Bueber ◽  
Henri G. Franquelim ◽  
Pierre Stegemann ◽  
...  
Keyword(s):  
Single Molecule ◽  
Signal Transmission ◽  
Resonance Energy Transfer ◽  
Dna Nanotechnology ◽  
Resonance Energy ◽  
Dna Origami ◽  
Potential Range ◽  
Voltage Sensing ◽  
Dynamics Simulations ◽  
Red Dye

Signal transmission in neurons goes along with changes in the transmembrane potential. To report them, different approaches including optical voltage-sensing dyes and genetically encoded voltage indicators have evolved. Here, we present a DNA nanotechnology-based system. Using DNA origami, we incorporate and optimize different properties such as membrane targeting and voltage sensing modularly. As a sensing unit, we use a hydrophobic red dye anchored to the membrane and an anionic green dye at the DNA connecting the DNA origami and the membrane dye anchor. Voltage-induced displacement of the anionic donor unit is read out by changes of Fluorescence Resonance Energy Transfer (FRET) of single sensors attached to liposomes. They show a FRET change of ~5% for ΔΨ=100 mV and allow adapting the potential range of highest sensitivity. Further, the working mechanism is rationalized by molecular dynamics simulations. Our approach holds potential for the application as non-genetically encoded sensors at membranes.

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.

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