scholarly journals Linking function to global and local dynamics in an elevator-type transporter

2021 ◽  
Vol 118 (49) ◽  
pp. e2025520118
Author(s):  
Didar Ciftci ◽  
Chloe Martens ◽  
Vishnu G. Ghani ◽  
Scott C. Blanchard ◽  
Argyris Politis ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Structural Changes ◽  
Substrate Affinity ◽  
Substrate Uptake ◽  
Local Dynamics ◽  
Transport Efficiency ◽  
Temporal Relationships ◽  
And Function ◽  
Global And Local

Transporters cycle through large structural changes to translocate molecules across biological membranes. The temporal relationships between these changes and function, and the molecular properties setting their rates, determine transport efficiency—yet remain mostly unknown. Using single-molecule fluorescence microscopy, we compare the timing of conformational transitions and substrate uptake in the elevator-type transporter GltPh. We show that the elevator-like movements of the substrate-loaded transport domain across membranes and substrate release are kinetically heterogeneous, with rates varying by orders of magnitude between individual molecules. Mutations increasing the frequency of elevator transitions and reducing substrate affinity diminish transport rate heterogeneities and boost transport efficiency. Hydrogen deuterium exchange coupled to mass spectrometry reveals destabilization of secondary structure around the substrate-binding site, suggesting that increased local dynamics leads to faster rates of global conformational changes and confers gain-of-function properties that set transport rates.

Influence of Ascorbic Acid on the Structure and Function of Alpha-2- macroglobulin: Investigations using Spectroscopic and Thermodynamic Techniques

2020 ◽  
Vol 27 (3) ◽  
pp. 201-209
Author(s):  
Syed Saqib Ali ◽  
Mohammad Khalid Zia ◽  
Tooba Siddiqui ◽  
Haseeb Ahsan ◽  
Fahim Halim Khan
Keyword(s):  
Ascorbic Acid ◽  
Conformational Changes ◽  
Structural Changes ◽  
The Body ◽  
Titration Calorimetry ◽  
Spectroscopic Techniques ◽  
Human Beings ◽  
Plasma Ascorbic Acid ◽  
Dietary Antioxidant ◽  
And Function

Background: Ascorbic acid is a classic dietary antioxidant which plays an important role in the body of human beings. It is commonly found in various foods as well as taken as dietary supplement. Objective: The plasma ascorbic acid concentration may range from low, as in chronic or acute oxidative stress to high if delivered intravenously during cancer treatment. Sheep alpha-2- macroglobulin (α2M), a human α2M homologue is a large tetrameric glycoprotein of 630 kDa with antiproteinase activity, found in sheep’s blood. Methods: In the present study, the interaction of ascorbic acid with alpha-2-macroglobulin was explored in the presence of visible light by utilizing various spectroscopic techniques and isothermal titration calorimetry (ITC). Results: UV-vis and fluorescence spectroscopy suggests the formation of a complex between ascorbic acid and α2M apparent by increased absorbance and decreased fluorescence. Secondary structural changes in the α2M were investigated by CD and FT-IR spectroscopy. Our findings suggest the induction of subtle conformational changes in α2M induced by ascorbic acid. Thermodynamics signatures of ascorbic acid and α2M interaction indicate that the binding is an enthalpy-driven process. Conclusion: It is possible that ascorbic acid binds and compromises antiproteinase activity of α2M by inducing changes in the secondary structure of the protein.

scholarly journals NCIPLOT4: A New Step Towards a Fast Quantification of Noncovalent Interactions

2019 ◽  
Author(s):  
Roberto Boto ◽  
Francesca Peccati ◽  
Rubén Laplaza ◽  
chaoyu quan ◽  
Alessandra Carbone ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Driving Forces ◽  
Noncovalent Interactions ◽  
Noncovalent Interaction ◽  
Simple Approach ◽  
Machine Learning Algorithms ◽  
Definition Of ◽  
And Function ◽  
The Relationship

<br>The quantification of noncovalent interactions in big systems is of crucial importance for understanding the structure and function of biosystems. The NCI method [J. Am. Chem. Soc. 132 , 6498 (2010)] enables to identify attractive and repulsive noncovalent interactions from promolecular densities in a fast manner. However, the approach remained up to now visual/qualitative, the relationship with energetics was conspicuously missing. We present a new version of NCIPLOT which allows quantifying the properties of the NonCovalent Interaction (NCI) regions in a fast manner. In order to do so, the definition of NCI volumes is introduced, which allows quantification of intra and intermolecular NCI properties in big systems where wavefunctions are not available. The connection between these integrals and energetics is reviewed for benchmark systems (S66 8), showing that our simple approach can lead to GGAquality energies while scaling with the number of atoms involved in the interaction (not the total number of atoms). The new implementation also includes an adaptive grid which allows the computation in a fast, parallelizable and efficient computational environment. The relationship with energetics derived from force fields is highlighted<br>and the faster algorithm exploited to analyze the evolution of interactions along MD trajectories. Through machine learning algorithms we characterize the relevance of NCI integrals in understanding the energetics of big systems, which is then applied in revealing the energetic changes along conformational changes, as well as identifying the atoms involved. This simple approach enables to identify the driving forces in biomolecular structural changes both at the spatial and energetic levels, while going beyond a mere parametrized-distances analysis.<br>

Nanoscale Analysis of the Effect of Pathogenic Mutations on Polycystin-1

2010 ◽  
Author(s):  
Liang Ma ◽  
Meixiang Xu ◽  
Andres F. Oberhauser
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Structure And Function ◽  
Eukaryotic Cells ◽  
Mechanical Forces ◽  
Force Microscopy ◽  
Physiological Conditions ◽  
Atomic Force ◽  
And Function

The activity of proteins and their complexes often involves the conversion of chemical energy (stored or supplied) into mechanical work through conformational changes. Mechanical forces are also crucial for the regulation of the structure and function of cells and tissues. Thus, the shape of eukaryotic cells is the result of cycles of mechano-sensing, mechano-transduction, and mechano-response. Recently developed single-molecule atomic force microscopy (AFM) techniques can be used to manipulate single molecules, both in real time and under physiological conditions, and are ideally suited to directly quantify the forces involved in both intra- and intermolecular protein interactions. In combination with molecular biology and computer simulations, these techniques have been applied to characterize the unfolding and refolding reactions in a variety of proteins, such as titin (an elastic mechano-sensing protein found in muscle) and polycystin-1 (PC1, a mechanosensor found in the kidney).

NCIPLOT4: A New Step Towards a Fast Quantification of Noncovalent Interactions

2020 ◽  
Author(s):  
Roberto Boto ◽  
Francesca Peccati ◽  
Rubén Laplaza ◽  
chaoyu quan ◽  
Alessandra Carbone ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Driving Forces ◽  
Noncovalent Interactions ◽  
Noncovalent Interaction ◽  
Simple Approach ◽  
Machine Learning Algorithms ◽  
Definition Of ◽  
And Function ◽  
The Relationship

<br>The quantification of noncovalent interactions in big systems is of crucial importance for understanding the structure and function of biosystems. The NCI method [J. Am. Chem. Soc. 132 , 6498 (2010)] enables to identify attractive and repulsive noncovalent interactions from promolecular densities in a fast manner. However, the approach remained up to now visual/qualitative, the relationship with energetics was conspicuously missing. We present a new version of NCIPLOT which allows quantifying the properties of the NonCovalent Interaction (NCI) regions in a fast manner. In order to do so, the definition of NCI volumes is introduced, which allows quantification of intra and intermolecular NCI properties in big systems where wavefunctions are not available. The connection between these integrals and energetics is reviewed for benchmark systems (S66 8), showing that our simple approach can lead to GGAquality energies while scaling with the number of atoms involved in the interaction (not the total number of atoms). The new implementation also includes an adaptive grid which allows the computation in a fast, parallelizable and efficient computational environment. The relationship with energetics derived from force fields is highlighted<br>and the faster algorithm exploited to analyze the evolution of interactions along MD trajectories. Through machine learning algorithms we characterize the relevance of NCI integrals in understanding the energetics of big systems, which is then applied in revealing the energetic changes along conformational changes, as well as identifying the atoms involved. This simple approach enables to identify the driving forces in biomolecular structural changes both at the spatial and energetic levels, while going beyond a mere parametrized-distances analysis.<br>

scholarly journals Nanomechanics of the substrate binding domain of Hsp70 determine its allosteric ATP-induced conformational change

2017 ◽  
Vol 114 (23) ◽  
pp. 6040-6045 ◽  
Author(s):  
Soumit Sankar Mandal ◽  
Dale R. Merz ◽  
Maximilian Buchsteiner ◽  
Ruxandra I. Dima ◽  
Matthias Rief ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Structural Changes ◽  
Substrate Binding ◽  
Protein Structures ◽  
Binding Domain ◽  
Flexible Region ◽  
Structural Parts ◽  
The Individual ◽  
Substrate Binding Domain

Owing to the cooperativity of protein structures, it is often almost impossible to identify independent subunits, flexible regions, or hinges simply by visual inspection of static snapshots. Here, we use single-molecule force experiments and simulations to apply tension across the substrate binding domain (SBD) of heat shock protein 70 (Hsp70) to pinpoint mechanical units and flexible hinges. The SBD consists of two nanomechanical units matching 3D structural parts, called the α- and β-subdomain. We identified a flexible region within the rigid β-subdomain that gives way under load, thus opening up the α/β interface. In exactly this region, structural changes occur in the ATP-induced opening of Hsp70 to allow substrate exchange. Our results show that the SBD’s ability to undergo large conformational changes is already encoded by passive mechanics of the individual elements.

scholarly journals Does Covera-19 know ‘when to hold ‘em or ‘when to fold ‘em? A translational thought experiment

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Gerald Dieter Griffin
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Structural Integrity ◽  
Thought Experiment ◽  
Amino Acid Sequences ◽  
Acid And Base ◽  
Effects Of Ph ◽  
Base Balance ◽  
Environmental Milieu ◽  
And Function

AbstractThe function of proteins depends on their structure. The structural integrity of proteins is dynamic and depends on interacting nearby neighboring moieties that influence their properties and induce folding and structural changes. The conformational changes induced by these nearby neighbors in the micro-environmental milieu at that moment are guided by chemical or electrical bonding attractions.There are few literature references that describe the potential for environmental milieu changes to disfavor SARS-CoV-2 attachment to a receptor for survival outside of a host. There are many studies on the effects of pH (acid and base balance) supporting its importance for protein structure and function, but few focus on pH role in extracellular or intracellular protein or actionable requirements of Covera-19.‘Fold ‘em or Hold ‘em’ is seen by the various functions and effects of furin as it seeks an acidic milieu for action or compatible amino acid sequences which is currently aided by its histidine component and the structural changes of proteins as they enter or exit the host. Questions throughout the text are posed to focus on current thoughts as reviewing applicable COVID-19 translational research science in order to understand the complexities of Covid-19.The pH needs of COVID-19 players and its journey through the human host and environment as well as some efficacious readily available repurposed drugs and out-of-the box and easily available treatments are reviewed.

scholarly journals Molecular Dynamics of SARS-CoV-2 Nsp1 Structural Changes Associated with Variant Mutations

2021 ◽  
Author(s):  
Shokouh Rezaei ◽  
Yahya Sefidbakht ◽  
Filipe Pereira
Keyword(s):  
Molecular Dynamics ◽  
Conformational Changes ◽  
Structural Changes ◽  
Structure And Function ◽  
Dynamics Simulation ◽  
Structural Protein ◽  
Wild Type ◽  
Deletion Mutations ◽  
And Function

Abstract SARS-CoV-2 non-structural protein 1 (Nsp1) is a virulence factor that inhibits the translation of host mRNAs and interact with viral RNA. Despite the relevance of Nsp1, few studies have been conducted to understand the effect of mutations on Nsp1 structure and function. Here, we provide a molecular dynamics simulation of SARS-CoV-2 Nsp1, wild type and variants. We found that SARS-CoV-2 Nsp1 has a more Rg value than SARS-CoV-1 Nsp1, with indicate an effect on the folding protein. This result suggest that SARS-CoV-2 Nsp1 can more easily approach the active site of the ribosome compared to SARS-CoV-1 Nsp1. In addition, we found that the C-terminal of the SARS-CoV-2 Nsp1, in particular residues 164 to 170, are more flexible than other regions of SARS-CoV-2 Nsp1 and SARS-CoV-1 Nsp1, confirming the role of this region in the interaction with the 40S subunit. Moreover, multiple deletion mutations have been found in the N/C-terminal of the SARS-CoV-2 Nsp1, which seems the effect of SARS-CoV-2 Nsp1 multiple deletions is greater than that of substitutions. Among all deletions, D156-158 and D80-90 may destabilize the protein structure and possibly increase the virulence of the SARS-CoV-2. Overall, our findings reinforce the importance of studying Nsp1 conformational changes in new variants and its effect on virulence of SARS-CoV-2.

scholarly journals Two-colour single-molecule photoinduced electron transfer fluorescence imaging microscopy of chaperone dynamics

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jonathan Schubert ◽  
Andrea Schulze ◽  
Chrisostomos Prodromou ◽  
Hannes Neuweiler
Keyword(s):  
Electron Transfer ◽  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Conformational Changes ◽  
Photoinduced Electron Transfer ◽  
Structural Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescence Imaging Microscopy ◽  
Pet Probes

AbstractMany proteins are molecular machines, whose function is dependent on multiple conformational changes that are initiated and tightly controlled through biochemical stimuli. Their mechanistic understanding calls for spectroscopy that can probe simultaneously such structural coordinates. Here we present two-colour fluorescence microscopy in combination with photoinduced electron transfer (PET) probes as a method that simultaneously detects two structural coordinates in single protein molecules, one colour per coordinate. This contrasts with the commonly applied resonance energy transfer (FRET) technique that requires two colours per coordinate. We demonstrate the technique by directly and simultaneously observing three critical structural changes within the Hsp90 molecular chaperone machinery. Our results reveal synchronicity of conformational motions at remote sites during ATPase-driven closure of the Hsp90 molecular clamp, providing evidence for a cooperativity mechanism in the chaperone’s catalytic cycle. Single-molecule PET fluorescence microscopy opens up avenues in the multi-dimensional exploration of protein dynamics and allosteric mechanisms.

scholarly journals Kinetic and thermodynamic framework for P4-P6 RNA reveals tertiary motif modularity and modulation of the folding preferred pathway

2016 ◽  
Vol 113 (34) ◽  
pp. E4956-E4965 ◽  
Author(s):  
Namita Bisaria ◽  
Max Greenfeld ◽  
Charles Limouse ◽  
Dmitri S. Pavlichin ◽  
Hideo Mabuchi ◽  
...  
Keyword(s):  
Single Molecule ◽  
Structural Changes ◽  
Structural Information ◽  
Equilibrium Constants ◽  
Single Point ◽  
Single Point Mutation ◽  
Single Molecule Fret ◽  
Predictive Understanding ◽  
And Function ◽  
And Behavior

The past decade has seen a wealth of 3D structural information about complex structured RNAs and identification of functional intermediates. Nevertheless, developing a complete and predictive understanding of the folding and function of these RNAs in biology will require connection of individual rate and equilibrium constants to structural changes that occur in individual folding steps and further relating these steps to the properties and behavior of isolated, simplified systems. To accomplish these goals we used the considerable structural knowledge of the folded, unfolded, and intermediate states of P4-P6 RNA. We enumerated structural states and possible folding transitions and determined rate and equilibrium constants for the transitions between these states using single-molecule FRET with a series of mutant P4-P6 variants. Comparisons with simplified constructs containing an isolated tertiary contact suggest that a given tertiary interaction has a stereotyped rate for breaking that may help identify structural transitions within complex RNAs and simplify the prediction of folding kinetics and thermodynamics for structured RNAs from their parts. The preferred folding pathway involves initial formation of the proximal tertiary contact. However, this preference was only ∼10 fold and could be reversed by a single point mutation, indicating that a model akin to a protein-folding contact order model will not suffice to describe RNA folding. Instead, our results suggest a strong analogy with a modified RNA diffusion-collision model in which tertiary elements within preformed secondary structures collide, with the success of these collisions dependent on whether the tertiary elements are in their rare binding-competent conformations.

scholarly journals Observation of long-range tertiary interactions during ligand binding by the TPP riboswitch aptamer

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Van K Duesterberg ◽  
Irena T Fischer-Hwang ◽  
Christian F Perez ◽  
Daniel W Hogan ◽  
Steven M Block
Keyword(s):  
Ligand Binding ◽  
Long Range ◽  
Single Molecule ◽  
Conformational Changes ◽  
Structural Changes ◽  
Regulatory Element ◽  
Optical Trap ◽  
Resonance Energy ◽  
Thiamine Pyrophosphate ◽  
Tertiary Interactions

The thiamine pyrophosphate (TPP) riboswitch is a cis-regulatory element in mRNA that modifies gene expression in response to TPP concentration. Its specificity is dependent upon conformational changes that take place within its aptamer domain. Here, the role of tertiary interactions in ligand binding was studied at the single-molecule level by combined force spectroscopy and Förster resonance energy transfer (smFRET), using an optical trap equipped for simultaneous smFRET. The ‘Force-FRET’ approach directly probes secondary and tertiary structural changes during folding, including events associated with binding. Concurrent transitions observed in smFRET signals and RNA extension revealed differences in helix-arm orientation between two previously-identified ligand-binding states that had been undetectable by spectroscopy alone. Our results show that the weaker binding state is able to bind to TPP, but is unable to form a tertiary docking interaction that completes the binding process. Long-range tertiary interactions stabilize global riboswitch structure and confer increased ligand specificity.

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