scholarly journals Molecular fluctuations as a ruler of force-induced protein conformations

2020 ◽  
Author(s):  
Andrew Stannard ◽  
Marc Mora ◽  
Amy E.M. Beedle ◽  
Marta Castro-Lopez ◽  
Stephanie Board ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dynamic Equilibrium ◽  
Conformational Dynamics ◽  
Magnetic Tweezers ◽  
Polymer Physics ◽  
Variance Analysis ◽  
Contour Length ◽  
Stable States ◽  
Wide Range ◽  
Dynamics Simulations

Molecular fluctuations directly reflect the underlying energy landscape. Variance analysis can probe protein dynamics in several biochemistry-driven approaches, yet measurement of probe-independent fluctuations in proteins exposed to mechanical forces remains only accessible through steered molecular dynamics simulations. Using single molecule magnetic tweezers, here we conduct variance analysis to show that individual unfolding and refolding transitions occurring in dynamic equilibrium in a single protein under force are hallmarked by a change in the protein's end-to-end fluctuations, revealing a change in protein stiffness. By unfolding and refolding three structurally distinct proteins under a wide range of constant forces, we demonstrate that the associated change in protein compliance to reach force-induced thermodynamically-stable states scales with the protein's contour length, in agreement with the sequence-independent FJC model of polymer physics. Our findings will help probe the conformational dynamics of proteins exposed to mechanical force at high resolution, of central importance in mechanosensing and mechanotransduction.

scholarly journals Dynamic interactions of type I cohesin modules fine-tune the structure of the cellulosome ofClostridium thermocellum

2018 ◽  
Vol 115 (48) ◽  
pp. E11274-E11283 ◽  
Author(s):  
Anders Barth ◽  
Jelle Hendrix ◽  
Daniel Fried ◽  
Yoav Barak ◽  
Edward A. Bayer ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dynamic Equilibrium ◽  
Conformational Dynamics ◽  
Anaerobic Bacteria ◽  
Spatial Arrangement ◽  
Industrial Applications ◽  
Type I ◽  
Binding Modes ◽  
Dynamic Interactions ◽  
Dynamics Simulations

Efficient degradation of plant cell walls by selected anaerobic bacteria is performed by large extracellular multienzyme complexes termed cellulosomes. The spatial arrangement within the cellulosome is organized by a protein called scaffoldin, which recruits the cellulolytic subunits through interactions between cohesin modules on the scaffoldin and dockerin modules on the enzymes. Although many structural studies of the individual components of cellulosomal scaffoldins have been performed, the role of interactions between individual cohesin modules and the flexible linker regions between them are still not entirely understood. Here, we report single-molecule measurements using FRET to study the conformational dynamics of a bimodular cohesin segment of the scaffoldin protein CipA ofClostridium thermocellum. We observe compacted structures in solution that persist on the timescale of milliseconds. The compacted conformation is found to be in dynamic equilibrium with an extended state that shows distance fluctuations on the microsecond timescale. Shortening of the intercohesin linker does not destabilize the interactions but reduces the rate of contact formation. Upon addition of dockerin-containing enzymes, an extension of the flexible state is observed, but the cohesin–cohesin interactions persist. Using all-atom molecular-dynamics simulations of the system, we further identify possible intercohesin binding modes. Beyond the view of scaffoldin as “beads on a string,” we propose that cohesin–cohesin interactions are an important factor for the precise spatial arrangement of the enzymatic subunits in the cellulosome that leads to the high catalytic synergy in these assemblies and should be considered when designing cellulosomes for industrial applications.

scholarly journals Protein dynamics enables phosphorylation of buried residues in Cdk2/Cyclin A-bound p27

2020 ◽  
Author(s):  
João Henriques ◽  
Kresten Lindorff-Larsen
Keyword(s):  
Protein Phosphorylation ◽  
Protein Dynamics ◽  
Single Molecule ◽  
Protein Function ◽  
Cell Cycle Progression ◽  
Conformational Dynamics ◽  
Protein Structures ◽  
Cyclin A ◽  
General Role ◽  
Wide Range

AbstractProteins carry out a wide range of functions that are tightly regulated in space and time. Protein phosphorylation is the most common post-translation modification of proteins and plays key roles in the regulation of many biological processes. The finding that many phosphorylated residues are not solvent exposed in the unphosphorylated state opens several questions for understanding the mechanism that underlies phosphorylation and how phosphorylation may affect protein structures. First, since kinases need access to the phosphorylated residue, how do such buried residues become modified? Second, once phosphorylated, what are the structural effects of phosphorylation of buried residues and do they lead to changed conformational dynamics. We have used the ternary complex between p27, Cdk2 and Cyclin A to study these questions using enhanced sampling molecular dynamics simulations. In line with previous NMR and single-molecule fluorescence experiments we observe transient exposure of Tyr88 in p27, even in its unphosphorylated state. Once Tyr88 is phosphorylated, we observe a coupling to a second site, thus making Tyr74 more easily exposed, and thereby the target for a second phosphorylation step. Our observations provide atomic details on how protein dynamics plays a role in modulating multi-site phosphorylation in p27, thus supplementing previous experimental observations. More generally, we discuss how the observed phenomenon of transient exposure of buried residues may play a more general role in regulating protein function.Significance StatementProtein phosphorylation is a common post-translation modification and is carried out by kinases. While many phosphorylation sites are located in disordered regions of proteins or in loops, a surprisingly large number of modification sites are buried inside folded domains. This observation led us to ask the question of how kinases gain access to such buried residues. We used the complex between p27, a regulator of cell cycle progression, and Cyclin-dependent kinase 2/Cyclin A to study this problem. We hypothesized that transient exposure of buried tyrosines in p27 to the solvent would make them accessible to kinases, explaining how buried residues get modified. We provide an atomic-level description of these dynamic processes revealing how protein dynamics plays a role in regulation.

scholarly journals A flow extension tethered particle motion assay for single-molecule proteolysis

2019 ◽  
Author(s):  
Andrew A. Drabek ◽  
Joseph J. Loparo ◽  
Stephen C. Blacklow
Keyword(s):  
Single Molecule ◽  
Magnetic Force ◽  
Magnetic Tweezers ◽  
Signaling Proteins ◽  
Mechanical Tension ◽  
Tobacco Etch Virus Protease ◽  
Single Molecule Level ◽  
Wide Range ◽  
Tethered Particle Motion

AbstractRegulated proteolysis of signaling proteins under mechanical tension enables cells to communicate with their environment in a variety of developmental and physiologic contexts. The role of force in inducing proteolytic sensitivity has been explored using magnetic tweezers at the single-molecule level with bead-tethered assays, but such efforts have been limited by challenges in ensuring that beads are not restrained by multiple tethers. Here, we describe a multiplexed assay for single-molecule proteolysis that overcomes the multiple-tether problem using a flow extension (FLEX) strategy on a microscope equipped with magnetic tweezers. Particle tracking and computational sorting of flow-induced displacements allows assignment of tethered substrates into singly-captured and multiply-tethered bins, with the fraction of fully mobile, single-tethered substrates depending inversely on the concentration of substrate loaded on the coverslip. Computational exclusion of multiply-tethered beads enables robust assessment of on-target proteolysis by the highly specific tobacco etch virus protease and the more promiscuous metalloprotease ADAM17. This method should be generally applicable to a wide range of proteases and readily extensible to robust evaluation of proteolytic sensitivity as a function of applied magnetic force.

scholarly journals Drastic changes in conformational dynamics of the antiterminator M2-1 regulate transcription efficiency in Pneumovirinae

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Cedric Leyrat ◽  
Max Renner ◽  
Karl Harlos ◽  
Juha T Huiskonen ◽  
Jonathan M Grimes
Keyword(s):  
Zinc Finger ◽  
Rna Binding ◽  
Dynamic Equilibrium ◽  
Conformational Dynamics ◽  
Adenosine Monophosphate ◽  
Zinc Finger Domain ◽  
Rna Sequences ◽  
Solution Structures ◽  
Closed Conformation ◽  
Dynamics Simulations

The M2-1 protein of human metapneumovirus (HMPV) is a zinc-binding transcription antiterminator which is highly conserved among pneumoviruses. We report the structure of tetrameric HMPV M2-1. Each protomer features a N-terminal zinc finger domain and an α-helical tetramerization motif forming a rigid unit, followed by a flexible linker and an α-helical core domain. The tetramer is asymmetric, three of the protomers exhibiting a closed conformation, and one an open conformation. Molecular dynamics simulations and SAXS demonstrate a dynamic equilibrium between open and closed conformations in solution. Structures of adenosine monophosphate- and DNA- bound M2-1 establish the role of the zinc finger domain in base-specific recognition of RNA. Binding to ‘gene end’ RNA sequences stabilized the closed conformation of M2-1 leading to a drastic shift in the conformational landscape of M2-1. We propose a model for recognition of gene end signals and discuss the implications of these findings for transcriptional regulation in pneumoviruses.

scholarly journals Modular, ultra-stable, and highly parallel protein force spectroscopy in magnetic tweezers using peptide linkers

2018 ◽  
Author(s):  
Achim Löf ◽  
Philipp U Walker ◽  
Steffen M Sedlak ◽  
Tobias Obser ◽  
Maria A Brehm ◽  
...  
Keyword(s):  
Single Molecule ◽  
Force Spectroscopy ◽  
Magnetic Tweezers ◽  
Protein Domain ◽  
High Yield ◽  
Primary Hemostasis ◽  
Wide Range ◽  
And Function ◽  
Von Willebrand

Single-molecule force spectroscopy has provided unprecedented insights into protein folding, force-regulation, and function. Here, we present a modular magnetic tweezers force spectroscopy approach that uses elastin-like polypeptide linkers to provide a high yield of protein tethers. Our approach extends protein force spectroscopy into the low force (<1 pN) regime and enables ultra-stable measurements on many molecules in parallel. We present (un-)folding data for the single protein domain ddFLN4 and for the large multi-domain dimeric protein von Willebrand factor (VWF) that is critically involved in primary hemostasis. The measurements reveal exponential force-dependencies of unfolding and refolding rates, directly resolve the stabilization of the VWF A2 domain by Ca2+, and discover transitions in the VWF C-domain stem at low forces that likely constitute the first steps of VWF activation in vivo. Our modular attachment approach will enable precise and multiplexed force spectroscopy measurements for a wide range of proteins in the physiologically relevant force regime.

scholarly journals Insight into helicase mechanism and function revealed through single-molecule approaches

2010 ◽  
Vol 43 (2) ◽  
pp. 185-217 ◽  
Author(s):  
Jaya G. Yodh ◽  
Michael Schlierf ◽  
Taekjip Ha
Keyword(s):  
Single Molecule ◽  
Conformational Dynamics ◽  
Magnetic Tweezers ◽  
Repetitive Behaviors ◽  
Helicase Activity ◽  
Step Size ◽  
Mechanistic Insight ◽  
Combination Of Methods ◽  
And Function ◽  
Insight Into

AbstractHelicases are a class of nucleic acid (NA) motors that catalyze NTP-dependent unwinding of NA duplexes into single strands, a reaction essential to all areas of NA metabolism. In the last decade, single-molecule (sm) technology has proven to be highly useful in revealing mechanistic insight into helicase activity that is not always detectable via ensemble assays. A combination of methods based on fluorescence, optical and magnetic tweezers, and flow-induced DNA stretching has enabled the study of helicase conformational dynamics, force generation, step size, pausing, reversal and repetitive behaviors during translocation and unwinding by helicases working alone and as part of multiprotein complexes. The contributions of these sm investigations to our understanding of helicase mechanism and function will be discussed.

scholarly journals Probing conformational dynamics of an enzymatic active site by an in situ single fluorogenic probe under piconewton force manipulation

2016 ◽  
Vol 113 (52) ◽  
pp. 15006-15011 ◽  
Author(s):  
Nibedita Pal ◽  
Meiling Wu ◽  
H. Peter Lu
Keyword(s):  
Single Molecule ◽  
Active Site ◽  
Complex Dynamics ◽  
Conformational Dynamics ◽  
Enzymatic Reaction ◽  
Product Formation ◽  
Magnetic Tweezers ◽  
Complex Nature ◽  
Enzyme Substrate ◽  
Multiple Product

Unraveling the conformational details of an enzyme during the essential steps of a catalytic reaction (i.e., enzyme–substrate interaction, enzyme–substrate active complex formation, nascent product formation, and product release) is challenging due to the transient nature of intermediate conformational states, conformational fluctuations, and the associated complex dynamics. Here we report our study on the conformational dynamics of horseradish peroxidase using single-molecule multiparameter photon time-stamping spectroscopy with mechanical force manipulation, a newly developed single-molecule fluorescence imaging magnetic tweezers nanoscopic approach. A nascent-formed fluorogenic product molecule serves as a probe, perfectly fitting in the enzymatic reaction active site for probing the enzymatic conformational dynamics. Interestingly, the product releasing dynamics shows the complex conformational behavior with multiple product releasing pathways. However, under magnetic force manipulation, the complex nature of the multiple product releasing pathways disappears and more simplistic conformations of the active site are populated.

scholarly journals Inter-domain dynamics in the chaperone SurA and multi-site binding to its unfolded outer membrane protein clients

2019 ◽  
Author(s):  
Antonio N. Calabrese ◽  
Bob Schiffrin ◽  
Matthew Watson ◽  
Theodoros K. Karamanos ◽  
Martin Walko ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Single Molecule ◽  
Conformational Changes ◽  
Outer Membrane Protein ◽  
Conformational Dynamics ◽  
Core Domain ◽  
The Core ◽  
Single Molecule Fret ◽  
Dynamics Simulations

AbstractThe periplasmic chaperone SurA plays a key role in outer membrane protein (OMP) biogenesis. E. coli SurA comprises a core domain and two peptidylprolyl isomerase domains (P1 and P2), but how it binds its OMP clients and the mechanism(s) of its chaperone action remain unclear. Here, we have used chemical cross-linking, hydrogen-deuterium exchange, single-molecule FRET and molecular dynamics simulations to map the client binding site(s) on SurA and to interrogate the role of conformational dynamics of the chaperone’s domains in OMP recognition. We demonstrate that SurA samples a broad array of conformations in solution in which P2 primarily lies closer to the core/P1 domains than suggested by its crystal structure. Multiple binding sites for OMPs are located primarily in the core domain, with binding of the unfolded OMP resulting in conformational changes between the core/P1 domains. Together, the results portray a model in which unfolded OMP substrates bind in a cradle formed between the SurA domains, with structural flexibility between its domains assisting OMP recognition, binding and release.

scholarly journals Magnetic tweezers meets AFM: ultra-stable protein dynamics across the force spectrum

2021 ◽  
Author(s):  
Alvaro Alonso-Caballero ◽  
Rafael Tapia-Rojo ◽  
Carmen L. Badilla ◽  
Julio M. Fernandez
Keyword(s):  
Protein Dynamics ◽  
Single Molecule ◽  
Magnetic Tweezers ◽  
Spectroscopy Technique ◽  
Mechanical Loads ◽  
Force Microscopy ◽  
Adhesive Protein ◽  
Atomic Force ◽  
Dissipation Of Energy ◽  
Wide Range

Proteins that operate under force—cell adhesion, mechanosensing—exhibit a wide range of mechanostabilities. Single-molecule magnetic tweezers has enabled the exploration of the dynamics under force of these proteins with subpiconewton resolution and unbeatable stability in the 0.1-120 pN range. However, proteins featuring a high mechanostability (>120 pN) have remained elusive with this technique and have been addressed with Atomic Force Microscopy (AFM), which can reach higher forces but displays less stability and resolution. Herein, we develop a magnetic tweezers approach that can apply AFM-like mechanical loads while maintaining its hallmark resolution and stability in a range of forces that spans from 1 to 500 pN. We demonstrate our approach by exploring the folding and unfolding dynamics of the highly mechanostable adhesive protein FimA from the Gram-positive pathogen Actinomyces oris. FimA unfolds at loads >300 pN, while its folding occurs at forces <15 pN, producing a large dissipation of energy that could be crucial for the shock absorption of mechanical challenges during host invasion. Our novel magnetic tweezers approach entails an all-in-one force spectroscopy technique for protein dynamics studies across a broad spectrum of physiologically-relevant forces and timescales.

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