scholarly journals Ligand modulation of the conformational dynamics of the A2A adenosine receptor revealed by single-molecule fluorescence

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Dennis D. Fernandes ◽  
Chris Neale ◽  
Gregory-Neal W. Gomes ◽  
Yuchong Li ◽  
Aimen Malik ◽  
...  
Keyword(s):  
G Protein ◽  
Single Molecule ◽  
Global Analysis ◽  
Conformational Dynamics ◽  
Quantitative Description ◽  
Correlation Spectroscopy ◽  
Conformational Ensemble ◽  
Dynamic Quenching ◽  
Single Molecule Fluorescence ◽  
Inter State

AbstractG protein-coupled receptors (GPCRs) are the largest class of transmembrane proteins, making them an important target for therapeutics. Activation of these receptors is modulated by orthosteric ligands, which stabilize one or several states within a complex conformational ensemble. The intra- and inter-state dynamics, however, is not well documented. Here, we used single-molecule fluorescence to measure ligand-modulated conformational dynamics of the adenosine A2A receptor (A2AR) on nanosecond to millisecond timescales. Experiments were performed on detergent-purified A2R in either the ligand-free (apo) state, or when bound to an inverse, partial or full agonist ligand. Single-molecule Förster resonance energy transfer (smFRET) was performed on detergent-solubilized A2AR to resolve active and inactive states via the separation between transmembrane (TM) helices 4 and 6. The ligand-dependent changes of the smFRET distributions are consistent with conformational selection and with inter-state exchange lifetimes ≥ 3 ms. Local conformational dynamics around residue 2296.31 on TM6 was measured using fluorescence correlation spectroscopy (FCS), which captures dynamic quenching due to photoinduced electron transfer (PET) between a covalently-attached dye and proximal aromatic residues. Global analysis of PET-FCS data revealed fast (150–350 ns), intermediate (50–60 μs) and slow (200–300 μs) conformational dynamics in A2AR, with lifetimes and amplitudes modulated by ligands and a G-protein mimetic (mini-Gs). Most notably, the agonist binding and the coupling to mini-Gs accelerates and increases the relative contribution of the sub-microsecond phase. Molecular dynamics simulations identified three tyrosine residues (Y112, Y2887.53, and Y2907.55) as being responsible for the dynamic quenching observed by PET-FCS and revealed associated helical motions around residue 2296.31 on TM6. This study provides a quantitative description of conformational dynamics in A2AR and supports the idea that ligands bias not only GPCR conformations but also the dynamics within and between distinct conformational states of the receptor.

scholarly journals Ligand Modulation of the Conformational Dynamics of the A2A Adenosine Receptor Revealed by Single-Molecule Fluorescence

2020 ◽  
Author(s):  
Dennis D. Fernandes ◽  
Chris Neale ◽  
Gregory-Neal W. Gomes ◽  
Yuchong Li ◽  
Aimen Malik ◽  
...  
Keyword(s):  
G Protein ◽  
Single Molecule ◽  
Global Analysis ◽  
Conformational Dynamics ◽  
Quantitative Description ◽  
Correlation Spectroscopy ◽  
Conformational Ensemble ◽  
Dynamic Quenching ◽  
Single Molecule Fluorescence ◽  
Inter State

ABSTRACTG protein-coupled receptors (GPCRs) are the largest class of transmembrane proteins, making them an important target for therapeutics. Activation of these receptors is modulated by orthosteric ligands, which stabilize one or several states within a complex conformational ensemble. The intra-and inter-state dynamics, however, is not well documented. Here, we used single-molecule fluorescence to measure ligand-modulated conformational dynamics of the adenosine A2A Receptor (A2AR) on nanosecond to millisecond timescales. Experiments were performed on detergent-purified A2R in either the ligand-free (apo) state, or when bound to an inverse, partial or full agonist ligand. Single-molecule Förster resonance energy transfer (smFRET) was performed on detergent-solubilized A2AR to resolve active and inactive states via the separation between transmembrane (TM) helices 4 and 6. The ligand-dependent changes of the smFRET distributions are consistent with conformational selection and with inter-state exchange lifetimes ≥ 3 ms. Local conformational dynamics around residue 229 on TM6 was measured using Fluorescence Correlation Spectroscopy (FCS), which captures dynamic quenching due to photoinduced electron transfer (PET) between a covalently-attached dye and proximal aromatic residues. Global analysis of PET-FCS data revealed fast (150-350 ns), intermediate (50-60 μs) and slow (200-300 μs) conformational dynamics in A2AR, with lifetimes and amplitudes modulated by ligands and a G-protein mimetic (mini-Gs). Most notably, the agonist binding and the coupling to mini-Gs accelerates and increases the relative contribution of the sub-microsecond phase. Molecular dynamics simulations identified three tyrosine residues (Y112, Y288, and Y290) as being responsible for the dynamic quenching observed by PET-FCS and revealed associated helical motions around residue 229 on TM6. This study provides a quantitative description of conformational dynamics in A2AR and supports the idea that ligands bias not only GPCR conformations but also the dynamics within and between distinct conformational states of the receptor.

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

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.

scholarly journals Fast and Robust 2D Inverse Laplace Transformation of Single-Molecule Fluorescence Lifetime Data

2021 ◽  
Author(s):  
Saurabh Talele ◽  
John T. King
Keyword(s):  
Nmr Spectroscopy ◽  
Fluorescence Spectroscopy ◽  
Single Molecule ◽  
Fluorescence Lifetime ◽  
Conformational Dynamics ◽  
Correlation Spectroscopy ◽  
Great Promise ◽  
Biological Macromolecules ◽  
Single Molecule Fluorescence ◽  
Laplace Transformations

AbstractFluorescence spectroscopy at the single-molecule scale has been indispensable for studying conformational dynamics and rare states of biological macromolecules. Single-molecule 2D-fluorescence lifetime correlation spectroscopy (sm-2D-FLCS) is an emerging technique that holds great promise for the study of protein and nucleic acid dynamics as it 1) resolves conformational dynamics using a single chromophore, 2) measures forward and reverse transitions independently, and 3) has a dynamic window ranging from microseconds to seconds. However, the calculation of a 2D fluorescence relaxation spectrum requires an inverse Laplace transition (ILT), which is an ill-conditioned inversion that must be estimated numerically through a regularized minimization. The current methods for performing ILTs of fluorescence relaxation can be computationally inefficient, sensitive to noise corruption, and difficult to implement. Here, we adopt an approach developed for NMR spectroscopy (T1-T2 relaxometry) to perform 1D and 2D-ILTs on single-molecule fluorescence spectroscopy data using singular-valued decomposition and Tikhonov regularization. This approach provides fast, robust, and easy to implement Laplace inversions of single-molecule fluorescence data.Significance StatementInverse Laplace transformations are a powerful approach for analyzing relaxation data. The inversion computes a relaxation rate spectrum from experimentally measured temporal relaxation, circumventing the need to choose appropriate fitting functions. They are routinely performed in NMR spectroscopy and are becoming increasing used in single-molecule fluorescence experiments. However, as Laplace inversions are ill-conditioned transformations, they must be estimated from regularization algorithms that are often computationally costly and difficult to implement. In this work, we adopt an algorithm first developed for NMR relaxometry to provide fast, robust, and easy to implement 1D and 2D inverse Laplace transformations on single-molecule fluorescence data.

scholarly journals Allosteric modulation of the SARS-CoV-2 spike conformation

2021 ◽  
Author(s):  
Marco A Diaz-Salinas ◽  
Qi Li ◽  
Monir Ejemel ◽  
Yang Wang ◽  
James B Munro
Keyword(s):  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Structural Data ◽  
Correlation Spectroscopy ◽  
Host Cells ◽  
Parallel Solution ◽  
Real Time Analysis ◽  
Multiple Conformations

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells through binding to angiotensin-converting enzyme 2 (ACE2), which is mediated by the receptor-binding domain (RBD) of the viral spike (S) glycoprotein. Structural data and real-time analysis of conformational dynamics have shown that S can adopt multiple conformations, which mediate the exposure of the ACE2-binding site in the RBD. Here, using single-molecule Förster resonance energy transfer (smFRET) imaging we report the effects of ACE2 and antibody binding on the conformational dynamics of S from the Wuhan-1 strain and the B.1 variant (D614G). We found that antibodies that target diverse epitopes, including those distal to the RBD, stabilize the RBD in a position competent for ACE2 binding. Parallel solution-based binding experiments using fluorescence correlation spectroscopy (FCS) indicated antibody-mediated enhancement of ACE2 binding. These findings inform on novel strategies for therapeutic antibody cocktails.

scholarly journals Labeling of Proteins for Single-Molecule Fluorescence Spectroscopy

2020 ◽  
Author(s):  
Franziska Zosel ◽  
Andrea Holla ◽  
Benjamin Schuler
Keyword(s):  
Fluorescence Spectroscopy ◽  
Single Molecule ◽  
Intrinsically Disordered Proteins ◽  
Conformational Dynamics ◽  
Control Sample ◽  
Resonance Energy ◽  
Disordered Proteins ◽  
Single Molecule Fluorescence ◽  
Intrinsically Disordered ◽  
Single Molecule Fluorescence Spectroscopy

Single-molecule fluorescence spectroscopy has become an important technique for studying the conformational dynamics and folding of proteins. A key step for performing such experiments is the availability of high-quality samples. Here we describe the practical details of a simple and widely applicable strategy for preparing proteins that are site-specifically labeled with a donor and an acceptor dye for single-molecule Förster resonance energy transfer (FRET) experiments. The method is based on introducing two cysteine residues that are labeled with maleimide-functionalized fluorophores, combined with high-resolution chromatography. We discuss how to optimize site-specific labeling even in the absence of orthogonal coupling chemistry and present purification strategies that are suitable for samples ranging from intrinsically disordered proteins to large folded proteins. We also discuss common problems in protein labeling, how to avoid them, and how to stringently control sample quality.<br>

scholarly journals Intramolecular dynamics of single molecules in free diffusion

2017 ◽  
Author(s):  
Charles Limouse ◽  
Jason C. Bell ◽  
Colin J. Fuller ◽  
Aaron F. Straight ◽  
Hideo Mabuchi
Keyword(s):  
Single Molecule ◽  
Conformational Dynamics ◽  
Correlation Spectroscopy ◽  
Biomolecular Systems ◽  
Optimal Position ◽  
Biochemical Processes ◽  
Fluorescence Correlation ◽  
Dna Molecule ◽  
Feedback Scheme ◽  
The Impact

AbstractBiomolecular systems such as multiprotein complexes or biopolymers can span several tens to several hundreds of nanometers, but the dynamics of such “mesocale” molecules remain challenging to probe. We have developed a single-molecule technique that uses Tracking Fluorescence Correlation Spectroscopy (tFCS) to measure the conformation and dynamics of molecular assemblies specifically at the mesoscale level (~100-1000 nm). tFCS is non-perturbative, as molecules, which are tracked in real-time, are untethered and freely diffusing. To achieve sub-diffraction spatial resolution, we use a feedback scheme which allows us to maintain the molecule at an optimal position within the laser intensity gradient. We find that tFCS is sufficiently sensitive to measure the distance fluctuations between two sites within a DNA molecule separated by distances as short as 1000 bp. We demonstrate that tFCS detects changes in the compaction of reconstituted chromatin, and can assay transient protein mediated interactions between distant sites in an individual DNA molecule. Our measurements highlight the impact that tFCS can have in the study of a wide variety of biochemical processes involving mesoscale conformational dynamics.

scholarly journals Conformational dynamics of Cas9 governing DNA cleavage revealed by single molecule FRET

2017 ◽  
Author(s):  
Mengyi Yang ◽  
Sijia Peng ◽  
Ruirui Sun ◽  
Jingdi Lin ◽  
Nan Wang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Cleavage ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Nuclease Activity ◽  
Single Molecule Fluorescence ◽  
Single Molecule Fret ◽  
Allosteric Communication ◽  
Target Binding

SummaryOff-target binding and cleavage by Cas9 pose as major challenges in its applications. How conformational dynamics of Cas9 governs its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms all spontaneously transits between three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We furthermore uncovered a surprising long-range allosteric communication between the HNH domain and RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox.

Sign in / Sign up

Export Citation Format

Share Document