Probing and Manipulating Enzyme Activity and Conformational Dynamics by Single-Molecule Afm-FRET and Magnetic Tweezers-FRET Ultramicroscopy

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.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1613404114 ◽

2016 ◽

Vol 113 (52) ◽

pp. 15006-15011 ◽

Cited By ~ 10

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.

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Molecular fluctuations as a ruler of force-induced protein conformations

10.1101/2020.12.18.423481 ◽

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.

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Monitoring the Structural Dynamics of U2 snRNA Via Single-Molecule Förster Resonance Energy Transfer

10.31237/osf.io/9j8gq ◽

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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Single-Molecule Conformational Dynamics of a Biologically Functional Hydroxocobalamin Riboswitch

Journal of the American Chemical Society ◽

10.1021/ja5076184 ◽

2014 ◽

Vol 136 (48) ◽

pp. 16832-16843 ◽

Cited By ~ 20

Author(s):

Erik D. Holmstrom ◽

Jacob T. Polaski ◽

Robert T. Batey ◽

David J. Nesbitt

Keyword(s):

Single Molecule ◽

Conformational Dynamics

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EXPRESS: Single-Molecule Fluorescence Techniques for Membrane Protein Dynamics Analysis

Applied Spectroscopy ◽

10.1177/00037028211009973 ◽

2021 ◽

pp. 000370282110099

Author(s):

Ziyu Yang ◽

Haiqi Xu ◽

Jiayu Wang ◽

Wei Chen ◽

Meiping Zhao

Keyword(s):

Membrane Protein ◽

Protein Dynamics ◽

Single Molecule ◽

Conformational Dynamics ◽

Correlation Spectroscopy ◽

Membrane Receptors ◽

Biological Macromolecules ◽

Dynamics Analysis ◽

Single Molecule Fluorescence ◽

Membrane Protein Dynamics

Fluorescence-based single molecule techniques, mainly including fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence resonance energy transfer (smFRET), are able to analyze the conformational dynamics and diversity of biological macromolecules. They have been applied to analysis of the dynamics of membrane proteins, such as membrane receptors and membrane transport proteins, due to their superior ability in resolving spatio-temporal heterogeneity and the demand of trace amounts of analytes. In this review, we first introduced the basic principle involved in FCS and smFRET. Then we summarized the labelling and immobilization strategies of membrane protein molecules, the confocal-based and TIRF-based instrumental configuration, and the data processing methods. The applications to membrane protein dynamics analysis are described in detail with the focus on how to select suitable fluorophores, labelling sites, experimental setup and analysis methods. In the last part, the remaining challenges to be addressed and further development in this field are also briefly discussed.

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Conformational entropy of a single peptide controlled under force governs protease recognition and catalysis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1803872115 ◽

2018 ◽

Vol 115 (45) ◽

pp. 11525-11530 ◽

Cited By ~ 4

Author(s):

Marcelo E. Guerin ◽

Guillaume Stirnemann ◽

David Giganti

Keyword(s):

Single Molecule ◽

Conformational Dynamics ◽

Conformational Sampling ◽

Enzymatic Reactions ◽

Sequence Specificity ◽

Proteomics Data ◽

Conformational Entropy ◽

Substrate Peptide ◽

The Impact

An immense repertoire of protein chemical modifications catalyzed by enzymes is available as proteomics data. Quantifying the impact of the conformational dynamics of the modified peptide remains challenging to understand the decisive kinetics and amino acid sequence specificity of these enzymatic reactions in vivo, because the target peptide must be disordered to accommodate the specific enzyme-binding site. Here, we were able to control the conformation of a single-molecule peptide chain by applying mechanical force to activate and monitor its specific cleavage by a model protease. We found that the conformational entropy impacts the reaction in two distinct ways. First, the flexibility and accessibility of the substrate peptide greatly increase upon mechanical unfolding. Second, the conformational sampling of the disordered peptide drives the specific recognition, revealing force-dependent reaction kinetics. These results support a mechanism of peptide recognition based on conformational selection from an ensemble that we were able to quantify with a torsional free-energy model. Our approach can be used to predict how entropy affects site-specific modifications of proteins and prompts conformational and mechanical selectivity.

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