scholarly journals Single molecule binding of a ligand to a G-protein-coupled receptor in real time using fluorescence correlation spectroscopy, rendered possible by nano-encapsulation in styrene maleic acid lipid particles

Nanoscale ◽  
2020 ◽  
Vol 12 (21) ◽  
pp. 11518-11525 ◽  
Author(s):  
Rachael L. Grime ◽  
Joelle Goulding ◽  
Romez Uddin ◽  
Leigh A. Stoddart ◽  
Stephen J. Hill ◽  
...  
Keyword(s):  
Real Time ◽  
G Protein ◽  
Single Molecule ◽  
Maleic Acid ◽  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
G Protein Coupled Receptor ◽  
Fluorescence Correlation ◽  
Lipid Particles ◽  
G Protein Coupled

Combining the technologies of encapsulation of GPCRs in SMA lipid particles with fluorescence correlation spectroscopy provides a versatile characterisation platform.

scholarly journals The use of fluorescence correlation spectroscopy to characterize the molecular mobility of fluorescently labelled G protein-coupled receptors

2016 ◽  
Vol 44 (2) ◽  
pp. 624-629 ◽  
Author(s):  
Laura E. Kilpatrick ◽  
Stephen J. Hill
Keyword(s):  
G Protein ◽  
Fluorescence Correlation Spectroscopy ◽  
Photon Counting ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
G Protein Coupled Receptors ◽  
Fluorescence Correlation ◽  
Membrane Bound ◽  
Molecular Brightness ◽  
G Protein Coupled

The membranes of living cells have been shown to be highly organized into distinct microdomains, which has spatial and temporal consequences for the interaction of membrane bound receptors and their signalling partners as complexes. Fluorescence correlation spectroscopy (FCS) is a technique with single cell sensitivity that sheds light on the molecular dynamics of fluorescently labelled receptors, ligands or signalling complexes within small plasma membrane regions of living cells. This review provides an overview of the use of FCS to probe the real time quantification of the diffusion and concentration of G protein-coupled receptors (GPCRs), primarily to gain insights into ligand–receptor interactions and the molecular composition of signalling complexes. In addition we document the use of photon counting histogram (PCH) analysis to investigate how changes in molecular brightness (ε) can be a sensitive indicator of changes in molecular mass of fluorescently labelled moieties.

The use of fluorescence correlation spectroscopy to characterise the molecular mobility of G protein-coupled receptors in membrane microdomains: an update

2021 ◽  
Vol 49 (4) ◽  
pp. 1547-1554
Author(s):  
Laura E. Kilpatrick ◽  
Stephen J. Hill
Keyword(s):  
G Protein ◽  
Fluorescence Correlation Spectroscopy ◽  
Molecular Mechanisms ◽  
Short Review ◽  
Correlation Spectroscopy ◽  
G Protein Coupled Receptors ◽  
Membrane Microdomains ◽  
Sensitivity Threshold ◽  
Fluorescence Correlation ◽  
G Protein Coupled

It has become increasingly apparent that some G protein-coupled receptors (GPCRs) are not homogeneously expressed within the plasma membrane but may instead be organised within distinct signalling microdomains. These microdomains localise GPCRs in close proximity with other membrane proteins and intracellular signalling partners and could have profound implications for the spatial and temporal control of downstream signalling. In order to probe the molecular mechanisms that govern GPCR pharmacology within these domains, fluorescence techniques with effective single receptor sensitivity are required. Of these, fluorescence correlation spectroscopy (FCS) is a technique that meets this sensitivity threshold. This short review will provide an update of the recent uses of FCS based techniques in conjunction with GPCR subtype selective fluorescent ligands to characterise dynamic ligand–receptor interactions in whole cells and using purified GPCRs.

scholarly journals The design of fluorescence correlation spectroscopy for single molecule techniques

2019 ◽  
Author(s):  
◽  
Zenia Norman
Keyword(s):  
Protein Synthesis ◽  
Real Time ◽  
Single Molecule ◽  
Fluorescence Correlation Spectroscopy ◽  
Ribosomal Proteins ◽  
High Speed ◽  
Correlation Spectroscopy ◽  
Experimental Conditions ◽  
High Count ◽  
Fluorescence Correlation

The ribosome is responsible for protein synthesis--a critical process in creating essential proteins for cell survival. Though bulk techniques yield valuable results about the structure of the ribosome, bulk techniques are not ideal in examining ribosomal dynamics and understanding kinetics involved in protein synthesis. On the contrary, the single molecule spectroscopy techniques are ideal in investigating the mechanisms of ribosome dynamics in real-time under equilibrium conditions. The latest advances in single molecule biophysics have opened numerous opportunities in the biological world to study the dynamics of molecules in real-time. Single molecule techniques such as Fluorescence Correlation Spectroscopy (FCS) have opened opportunities to study the ribosome as well as other ribosomal proteins. FCS is used to investigate diffusion coefficients, concentration, and kinetics of biological samples. In this thesis, we address the process of assembling an FCS system, as well as the design of a high-speed correlator. The high-speed correlator allows the software to handle the high data counts that can occur in single molecule experiments. A field-programmable gate arrays (FPGA) dual correlator and a multi-tau correlator are designed to handle the noise and high count rates that can dominate the signal. Also addressed in this thesis are ideal experimental conditions for successfully obtaining and troubleshooting results received from FCS curves.

Fluorescence correlation spectroscopy: The technique and its applications in soft matter

2018 ◽  
Vol 4 (4) ◽  
Author(s):  
Anjali Gupta ◽  
Jagadish Sankaran ◽  
Thorsten Wohland
Keyword(s):  
Single Molecule ◽  
Fluorescence Correlation Spectroscopy ◽  
Soft Matter ◽  
Photophysical Properties ◽  
Spatiotemporal Analysis ◽  
Correlation Spectroscopy ◽  
Basic Principles ◽  
Chemical Reaction Kinetics ◽  
Fluorescence Correlation ◽  
Wide Range

Abstract Fluorescence correlation spectroscopy (FCS) is a well-established single-molecule method used for the quantitative spatiotemporal analysis of dynamic processes in a wide range of samples. It possesses single-molecule sensitivity but provides ensemble averaged molecular parameters such as mobility, concentration, chemical reaction kinetics, photophysical properties and interaction properties. These parameters have been utilized to characterize a variety of soft matter systems. This review provides an overview of the basic principles of various FCS modalities, their instrumentation, data analysis, and the applications of FCS to soft matter systems.

Sign in / Sign up

Export Citation Format

Share Document