scholarly journals The P1 and P2 helices of the Guanidinium-II riboswitch interact in a ligand-dependent manner

2021 ◽  
Author(s):  
Christin Fuks ◽  
Sebastian Falkner ◽  
Nadine Schwierz ◽  
Martin Hengesbach
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Environmental Conditions ◽  
Structural Information ◽  
Coarse Grained ◽  
Regulation Mechanism ◽  
Dependent Manner ◽  
Loop Formation ◽  
Coarse Grained Simulations ◽  
Kissing Loop

ABSTRACTRiboswitch RNAs regulate gene expression by conformational changes induced by environmental conditions and specific ligand binding. The guanidine-II riboswitch is proposed to bind the small molecule guanidinium and to subsequently form a kissing loop interaction between the P1 and P2 hairpins. While an interaction was shown for isolated hairpins in crystallization and EPR experiments, an intrastrand kissing loop formation has not been demonstrated. Here, we report the first evidence of this interaction in cis in a ligand and Mg2+ dependent manner. Using single-molecule FRET spectroscopy and detailed structural information from coarse-grained simulations, we observe and characterize three interconvertible states representing an open and kissing loop conformation as well as a novel Mg2+ dependent state for the guanidine-II riboswitch from E. coli. The results further substantiate the proposed switching mechanism and provide detailed insight into the regulation mechanism for the guanidine-II riboswitch class. Combining single molecule experiments and coarse-grained simulations therefore provides a promising perspective in resolving the conformational changes induced by environmental conditions and to yield molecular insights into RNA regulation.

scholarly journals Simulating the function of sodium/proton antiporters

2015 ◽  
Vol 112 (40) ◽  
pp. 12378-12383 ◽  
Author(s):  
Raphael Alhadeff ◽  
Arieh Warshel
Keyword(s):  
Conformational Changes ◽  
Molecular Basis ◽  
Molecular Level ◽  
Structural Information ◽  
Coarse Grained ◽  
Charged State ◽  
Transport Models ◽  
Coarse Grained Model ◽  
Effective Transport ◽  
Molecular Origin

The molecular basis of the function of transporters is a problem of significant importance, and the emerging structural information has not yet been converted to a full understanding of the corresponding function. This work explores the molecular origin of the function of the bacterial Na+/H+ antiporter NhaA by evaluating the energetics of the Na+ and H+ movement and then using the resulting landscape in Monte Carlo simulations that examine two transport models and explore which model can reproduce the relevant experimental results. The simulations reproduce the observed transport features by a relatively simple model that relates the protein structure to its transporting function. Focusing on the two key aspartic acid residues of NhaA, D163 and D164, shows that the fully charged state acts as an Na+ trap and that the fully protonated one poses an energetic barrier that blocks the transport of Na+. By alternating between the former and latter states, mediated by the partially protonated protein, protons, and Na+ can be exchanged across the membrane at 2:1 stoichiometry. Our study provides a numerical validation of the need of large conformational changes for effective transport. Furthermore, we also yield a reasonable explanation for the observation that some mammalian transporters have 1:1 stoichiometry. The present coarse-grained model can provide a general way for exploring the function of transporters on a molecular level.

scholarly journals Nucleotides regulate the mechanical hierarchy between subdomains of the nucleotide binding domain of the Hsp70 chaperone DnaK

2015 ◽  
Vol 112 (33) ◽  
pp. 10389-10394 ◽  
Author(s):  
Daniela Bauer ◽  
Dale R. Merz ◽  
Benjamin Pelz ◽  
Kelly E. Theisen ◽  
Gail Yacyshyn ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Single Molecule ◽  
Conformational Changes ◽  
Mechanical Stability ◽  
Nucleotide Binding ◽  
Binding Domain ◽  
Coarse Grained ◽  
Nucleotide Binding Domain ◽  
Chaperone Dnak ◽  
Hsp70 Chaperone

The regulation of protein function through ligand-induced conformational changes is crucial for many signal transduction processes. The binding of a ligand alters the delicate energy balance within the protein structure, eventually leading to such conformational changes. In this study, we elucidate the energetic and mechanical changes within the subdomains of the nucleotide binding domain (NBD) of the heat shock protein of 70 kDa (Hsp70) chaperone DnaK upon nucleotide binding. In an integrated approach using single molecule optical tweezer experiments, loop insertions, and steered coarse-grained molecular simulations, we find that the C-terminal helix of the NBD is the major determinant of mechanical stability, acting as a glue between the two lobes. After helix unraveling, the relative stability of the two separated lobes is regulated by ATP/ADP binding. We find that the nucleotide stays strongly bound to lobe II, thus reversing the mechanical hierarchy between the two lobes. Our results offer general insights into the nucleotide-induced signal transduction within members of the actin/sugar kinase superfamily.

scholarly journals Salt Effects on the Thermodynamics of a Frameshifting RNA Pseudoknot under Tension

2016 ◽  
Author(s):  
Naoto Hori ◽  
Natalia A. Denesyuk ◽  
D. Thirumalai
Keyword(s):  
Single Molecule ◽  
Interaction Coefficient ◽  
Critical Force ◽  
Coarse Grained ◽  
Stem Loop ◽  
Ribosomal Frameshifting ◽  
Dna Hairpins ◽  
Coarse Grained Simulations ◽  
The Stability ◽  
Rna Interaction

Because of the potential link between -1 programmed ribosomal frameshifting and response of a pseudoknot (PK) RNA to force, a number of single molecule pulling experiments have been performed on PKs to decipher the mechanism of programmed ribosomal frameshifting. Motivated in part by these experiments, we performed simulations using a coarse-grained model of RNA to describe the response of a PK over a range of mechanical forces (fs) and monovalent salt concentrations (Cs). The coarse-grained simulations quantitatively reproduce the multistep thermal melting observed in experiments, thus validating our model. The free energy changes obtained in simulations are in excellent agreement with experiments. By varying f and C, we calculated the phase diagram that shows a sequence of structural transitions, populating distinct intermediate states. As f and C are changed, the stem-loop tertiary interactions rupture first, followed by unfolding of the 3’-end hairpin (I⇌F). Finally, the 5’-end hairpin unravels, producing an extended state (E⇌I). A theoretical analysis of the phase boundaries shows that the critical force for rupture scales as (log Cm)α with α = 1 (0.5) for E⇌I (I⇌F) transition. This relation is used to obtain the preferential ion-RNA interaction coefficient, which can be quantitatively measured in single-molecule experiments, as done previously for DNA hairpins. A by-product of our work is the suggestion that the frameshift efficiency is likely determined by the stability of the 5’ end hairpin that the ribosome first encounters during translation.

scholarly journals Trapping On-Pathway Intermediates for Large Scale Conformational Changes with Coarse-Grained Simulations

2017 ◽  
Vol 112 (3) ◽  
pp. 485a
Author(s):  
Laura Orellana ◽  
Özge Yoluk ◽  
Oliver Carrillo ◽  
Modesto Orozco ◽  
Erik Lindahl
Keyword(s):  
Conformational Changes ◽  
Large Scale ◽  
Coarse Grained ◽  
Coarse Grained Simulations

scholarly journals Spotlighting motors and controls of single FoF1-ATP synthase

2013 ◽  
Vol 41 (5) ◽  
pp. 1219-1226 ◽  
Author(s):  
Michael Börsch ◽  
Thomas M. Duncan
Keyword(s):  
Escherichia Coli ◽  
Atp Synthase ◽  
Single Molecule ◽  
Conformational Changes ◽  
Structural Information ◽  
E Coli ◽  
Single Molecule Fret ◽  
Membrane Enzyme ◽  
Subunit Rotation ◽  
Fof1 Atp Synthase

Subunit rotation is the mechanochemical intermediate for the catalytic activity of the membrane enzyme FoF1-ATP synthase. smFRET (single-molecule FRET) studies have provided insights into the step sizes of the F1 and Fo motors, internal transient elastic energy storage and controls of the motors. To develop and interpret smFRET experiments, atomic structural information is required. The recent F1 structure of the Escherichia coli enzyme with the ϵ-subunit in an inhibitory conformation initiated a study for real-time monitoring of the conformational changes of ϵ. The present mini-review summarizes smFRET rotation experiments and previews new smFRET data on the conformational changes of the CTD (C-terminal domain) of ϵ in the E. coli enzyme.

scholarly journals Coarse-grained simulations of conformational changes in the multidrug efflux transporter AcrB

2017 ◽  
Vol 13 (10) ◽  
pp. 2006-2014 ◽  
Author(s):  
Yead Jewel ◽  
Jin Liu ◽  
Prashanta Dutta
Keyword(s):  
Conformational Changes ◽  
Transmembrane Domain ◽  
Coarse Grained ◽  
Efflux Transporter ◽  
Multidrug Efflux ◽  
Coarse Grained Simulations

Deprotonation of Asp408 in the transmembrane domain induces opening of the cleft and closing of the exit in the porter domain.

scholarly journals Structural insights into a dimeric Psb27-photosystem II complex from a cyanobacterium Thermosynechococcus vulcanus

2021 ◽  
Vol 118 (5) ◽  
pp. e2018053118
Author(s):  
Guoqiang Huang ◽  
Yanan Xiao ◽  
Xiong Pi ◽  
Liang Zhao ◽  
Qingjun Zhu ◽  
...  
Keyword(s):  
Photosystem Ii ◽  
Conformational Changes ◽  
Water Oxidation ◽  
Structural Information ◽  
Regulation Mechanism ◽  
Loop Region ◽  
Luminal Side ◽  
Assembly Factor ◽  
Pigment Protein Complex ◽  
Structural Insights

Photosystem II (PSII) is a multisubunit pigment-protein complex and catalyzes light-driven water oxidation, leading to the conversion of light energy into chemical energy and the release of molecular oxygen. Psb27 is a small thylakoid lumen-localized protein known to serve as an assembly factor for the biogenesis and repair of the PSII complex. The exact location and binding fashion of Psb27 in the intermediate PSII remain elusive. Here, we report the structure of a dimeric Psb27-PSII complex purified from a psbV deletion mutant (ΔPsbV) of the cyanobacterium Thermosynechococcus vulcanus, solved by cryo-electron microscopy. Our structure showed that Psb27 is associated with CP43 at the luminal side, with specific interactions formed between Helix 2 and Helix 3 of Psb27 and a loop region between Helix 3 and Helix 4 of CP43 (loop C) as well as the large, lumen-exposed and hydrophilic E-loop of CP43. The binding of Psb27 imposes some conflicts with the N-terminal region of PsbO and also induces some conformational changes in CP43, CP47, and D2. This makes PsbO unable to bind in the Psb27-PSII. Conformational changes also occurred in D1, PsbE, PsbF, and PsbZ; this, together with the conformational changes occurred in CP43, CP47, and D2, may prevent the binding of PsbU and induce dissociation of PsbJ. This structural information provides important insights into the regulation mechanism of Psb27 in the biogenesis and repair of PSII.

scholarly journals Elucidation of ion effects on the Thermodynamics of RNA Folding

2018 ◽  
Author(s):  
Natalia A. Denesyuk ◽  
D. Thirumalai
Keyword(s):  
Free Energy ◽  
Single Molecule ◽  
Conformational Changes ◽  
Electrostatic Interactions ◽  
Rna Folding ◽  
Coarse Grained ◽  
Significant Advance ◽  
Rna Molecules ◽  
Ion Effects ◽  
The Difference

AbstractHow ions affect RNA folding thermodynamics and kinetics is an important but a vexing problem that remains unsolved. Experiments have shown that the free energy change, ΔG(c), of RNA upon folding varies with the salt concentration (c) as, ΔG(c) = kc ln c + const, where the coefficient kc is proportional to the difference in the uptake of ions (ion preferential coefficient), ΔΓ, between the folded and unfolded states. We performed simulations of a coarse-grained model, by modeling electrostatic interactions implicitly and with explicit representation of ions, to elucidate the molecular underpinnings of the relationship between folding free energy and ion preferential coefficient. Without any input from experiments, the simulations quantitatively reproduce the heat capacity for the −1 frame shifting pseudoknot (PK) from Beet Western Yellow Virus, thus validating the model. We show that ΔG(c) calculated directly from ΔΓ varies linearly with ln c (c < 0.2M), for a hairpin and the PK, thus demonstrating a molecular link between the two quantities for RNA molecules that undergo substantial conformational changes during folding. Explicit ion simulations also show the linear dependence of ΔG(c) on ln c at all c with kc = 2kBT, except that ΔG(c) values are shifted by about 2 kcal/mol higher than experiments at all salt concentrations. The discrepancy is due to an underestimate the Γ values for both the folded and unfolded states, while giving accurate values for ΔΓ. The predictions for the salt dependence of ΔΓ are amenable to test using single molecule pulling experiments. Our simulations, representing a significant advance in quantitatively describing ion effects in RNA, show that the framework provided here can be used to obtain accurate thermodynamics of RNA folding.

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