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.

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.

Single-molecule FRET for Ultrasensitive Detection of Biomolecules

2014 ◽  
Vol 1 (1) ◽  
Author(s):  
Kun Yang ◽  
Yong Yang ◽  
Chun-yang Zhang
Keyword(s):  
Single Molecule ◽  
New Technologies ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Molecular Probes ◽  
Ultrasensitive Detection ◽  
Single Molecule Fret ◽  
Biological Reaction ◽  
Spatio Temporal ◽  
Detection Of Biomolecules

AbstractSingle-molecule Förster resonance energy transfer (sm- FRET) has been widely employed to detect biomarkers and to probe the structure and dynamics of biomolecules. By monitoring the biological reaction in a spatio-temporal manner, smFRET can reveal the transient intermediates of biological processes that cannot be obtained by conventional ensemble measurements. This review provides an overview of singlemolecule FRET and its applications in ultrasensitive detection of biomolecules, including the major techniques and the molecular probes used for smFRET as well as the biomedical applications of smFRET. Especially, the combination of sm- FRET with new technologies might expand its applications in clinical diagnosis and biomedical research

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 The structural heterogeneity of α-synuclein is governed by several distinct subpopulations with interconversion times slower than milliseconds

2020 ◽  
Author(s):  
Jiaxing Chen ◽  
Sofia Zaer ◽  
Paz Drori ◽  
Joanna Zamel ◽  
Khalil Joron ◽  
...  
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Intrinsically Disordered Protein ◽  
Structural Heterogeneity ◽  
Conformational Space ◽  
Conformational Ensemble ◽  
Time Resolved ◽  
Intrinsically Disordered ◽  
Transient Nature

AbstractThe intrinsically disordered protein, α-synuclein, implicated in synaptic vesicle homeostasis and neurotransmitter release, is also associated with several neurodegenerative diseases. The different roles of α-synuclein are characterized by distinct structural states (membrane-bound, dimer, tetramer, oligomer, and fibril), which are originated from its various monomeric conformations. The pathological states, determined by the ensemble of α-synuclein monomer conformations and dynamic pathways of interconversion between dominant states, remain elusive due to their transient nature. Here, we use inter-dye distance distributions from bulk time-resolved Förster resonance energy transfer as restraints in discrete molecular dynamics simulations to map the conformational space of the α-synuclein monomer. We further confirm the generated conformational ensemble in orthogonal experiments utilizing far-UV circular dichroism and cross-linking mass spectrometry. Single-molecule protein-induced fluorescence enhancement measurements show that within this conformational ensemble, some of the conformations of α-synuclein are surprisingly stable, exhibiting conformational transitions slower than milliseconds. Our comprehensive analysis of the conformational ensemble reveals essential structural properties and potential conformations that promote its various functions in membrane interaction or oligomer and fibril formation.

scholarly journals Multiplexed, bioorthogonal labeling of multicomponent, biomolecular complexes using genomically encoded, non-canonical amino acids

2019 ◽  
Author(s):  
Bijoy J. Desai ◽  
Ruben L. Gonzalez
Keyword(s):  
Amino Acids ◽  
Structural Biology ◽  
Structural Dynamics ◽  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Functional Studies ◽  
Bioorthogonal Labeling ◽  
Biomolecular Complexes ◽  
And Function

Stunning advances in the structural biology of multicomponent biomolecular complexes (MBCs) have ushered in an era of intense, structure-guided mechanistic and functional studies of these complexes. Nonetheless, existing methods to site-specifically conjugate MBCs with biochemical and biophysical labels are notoriously impracticable and/or significantly perturb MBC assembly and function. To overcome these limitations, we have developed a general, multiplexed method in which we genomically encode non-canonical amino acids (ncAAs) into multiple, structure-informed, individual sites within a target MBC; select for ncAA-containing MBC variants that assemble and function like the wildtype MBC; and site-specifically conjugate biochemical or biophysical labels to these ncAAs. As a proof-of-principle, we have used this method to generate unique single-molecule fluorescence resonance energy transfer (smFRET) signals reporting on ribosome structural dynamics that have thus far remained inaccessible to smFRET studies of translation.

scholarly journals Evolution of structural dynamics in bilobed proteins

2020 ◽  
Author(s):  
Giorgos Gouridis ◽  
Yusran A. Muthahari ◽  
Marijn de Boer ◽  
Konstantinos Tassis ◽  
Alexandra Tsirigotaki ◽  
...  
Keyword(s):  
Structural Dynamics ◽  
Protein Evolution ◽  
Single Molecule ◽  
Protein Function ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protein Domain ◽  
Divergent Evolution ◽  
Exchange Mass Spectrometry ◽  
Cytoplasmic Transport

AbstractNovel biophysical tools allow the structural dynamics of proteins, and the regulation of such dynamics by binding partners, to be explored in unprecedented detail. Although this has provided critical insights into protein function, the means by which structural dynamics direct protein evolution remains poorly understood. Here, we investigated how proteins with a bilobed structure, composed of two related domains from the type-II periplasmic binding protein domain family, have undergone divergent evolution leading to modification of their structural dynamics and function. We performed a structural analysis of ~600 bilobed proteins with a common primordial structural core, which we complemented with biophysical studies to explore the structural dynamics of selected examples by single-molecule Förster resonance energy transfer and Hydrogen-Deuterium exchange mass spectrometry. We show that evolutionary modifications of the structural core, largely at its termini, enables distinct structural dynamics, allowing the diversification of these proteins into transcription factors, enzymes, and extra-cytoplasmic transport-related proteins. Structural embellishments of the core created new interdomain interactions that stabilized structural states, reshaping the active site geometry, and ultimately, altered substrate specificity. Our findings reveal an as yet unrecognized mechanism for the emergence of functional promiscuity during long periods of protein evolution and are applicable to a large number of domain architectures.

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 Structural Asymmetry and Kinetic Limping of Single Rotary F-ATP Synthases

Molecules ◽  
2019 ◽  
Vol 24 (3) ◽  
pp. 504 ◽  
Author(s):  
Hendrik Sielaff ◽  
Seiga Yanagisawa ◽  
Wayne D. Frasch ◽  
Wolfgang Junge ◽  
Michael Börsch
Keyword(s):  
Single Molecule ◽  
Binding Sites ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescence Probes ◽  
Structural Asymmetry ◽  
Rotationally Symmetric ◽  
Flow Through ◽  
Atp Synthases

F-ATP synthases use proton flow through the FO domain to synthesize ATP in the F1 domain. In Escherichia coli, the enzyme consists of rotor subunits γεc10 and stator subunits (αβ)3δab2. Subunits c10 or (αβ)3 alone are rotationally symmetric. However, symmetry is broken by the b2 homodimer, which together with subunit δa, forms a single eccentric stalk connecting the membrane embedded FO domain with the soluble F1 domain, and the central rotating and curved stalk composed of subunit γε. Although each of the three catalytic binding sites in (αβ)3 catalyzes the same set of partial reactions in the time average, they might not be fully equivalent at any moment, because the structural symmetry is broken by contact with b2δ in F1 and with b2a in FO. We monitored the enzyme’s rotary progression during ATP hydrolysis by three single-molecule techniques: fluorescence video-microscopy with attached actin filaments, Förster resonance energy transfer between pairs of fluorescence probes, and a polarization assay using gold nanorods. We found that one dwell in the three-stepped rotary progression lasting longer than the other two by a factor of up to 1.6. This effect of the structural asymmetry is small due to the internal elastic coupling.

scholarly journals Evaluating PI3 Kinase Isoforms Using Transcreener™ ADP Assays

2008 ◽  
Vol 13 (6) ◽  
pp. 476-485 ◽  
Author(s):  
Tony A. Klink ◽  
Karen M. Kleman-Leyer ◽  
Andrew Kopp ◽  
Thane A. Westermeyer ◽  
Robert G. Lowery
Keyword(s):  
Atp Hydrolysis ◽  
Rank Order ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Adenosine Diphosphate ◽  
Time Resolved ◽  
Lipid Kinases ◽  
Screening Assays ◽  
Lipid Environment ◽  
Phosphoinositide 3 Kinase

Development of drugs targeting lipid kinases has been delayed by the lack of robust screening assays. Methods are needed that can accommodate the presentation of different acceptor substrates in the optimal lipid environment. The Transcreener™ ADP Assay relies on homogeneous immunodetection of adenosine diphosphate (ADP), using either fluorescence polarization (FP) or time-resolved fluorescence resonance energy transfer (TR-FRET) as a signal output. Detection of ADP—the invariant product of all kinase reactions—provides complete flexibility for varying lipid substrate parameters. The authors used this assay to optimize dispersal methods for C8 and C16 phosphatidylinositol 4,5 bisphosphate substrates and to assess the effects of chain length on the activity and inhibition of phosphoinositide-3-kinase (PI3K) isoforms. The nonphysiological C8 substrate supported the highest activity. Known inhibitors were profiled using both the FP- and TR-FRET-based assays, and there was excellent concordance ( r2 = 0.93) in the IC 50 values. The overall rank order of inhibitors was the same using the C8 and C16 substrates, except for minor deviations. Adenosine triphosphate (ATP) hydrolysis in the absence of substrate was detected with the PI3Kα isoform, and inhibitors affected PI3Kα intrinsic ATP hydrolysis activity similarly to lipid phosphorylation. ( Journal of Biomolecular Screening 2008:476-485)

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