Single Molecule Height Measurements on a Membrane Protein in Nanometer-Scale Phospholipid Bilayer Disks

Langmuir ◽  
2000 ◽  
Vol 16 (14) ◽  
pp. 5993-5997 ◽  
Author(s):  
Timothy H. Bayburt ◽  
Joseph W. Carlson ◽  
Stephen G. Sligar
Keyword(s):  
Membrane Protein ◽  
Single Molecule ◽  
Phospholipid Bilayer ◽  
Nanometer Scale

scholarly journals Applications of Single-Molecule Methods to Membrane Protein Folding Studies

2018 ◽  
Vol 430 (4) ◽  
pp. 424-437 ◽  
Author(s):  
Robert E. Jefferson ◽  
Duyoung Min ◽  
Karolina Corin ◽  
Jing Yang Wang ◽  
James U. Bowie
Keyword(s):  
Protein Folding ◽  
Membrane Protein ◽  
Single Molecule ◽  
Membrane Protein Folding

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 Resolving the Dynamic Non-Covalent Interaction inside Membrane Protein Channel by Single-Molecule Interaction Spectrum

2018 ◽  
Author(s):  
Meng-Yin Li ◽  
Yi-Lun Ying ◽  
Xi-Xin Fu ◽  
Jie Yu ◽  
Shao-Chuang Liu ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Single Molecule ◽  
Ionic Current ◽  
Md Simulations ◽  
Energy Spectra ◽  
Membrane Channels ◽  
Non Covalent Interactions ◽  
Protein Channels ◽  
Covalent Interactions ◽  
Molecule Interaction

Millions of years of evolution have produced membrane protein channels capable of efficiently moving ions across the cell membrane. The underlying fundamental mechanisms that facilitate these actions greatly contribute to the weak non-covalent interactions. However, uncovering these dynamic interactions and its synergic network effects still remains challenging in both experimental techniques and molecule dynamics (MD) simulations. Here, we present a rational strategy that combines MD simulations and frequency-energy spectroscopy to identify and quantify the role of non-covalent interactions in carrier transport through membrane protein channels, as encoded in traditional single channel recording or ionic current. We employed wild-type aerolysin transporting of methylcytosine and cytosine as a model to explore the dynamic ionic signatures with non-stationary and non-linear frequency analysis. Our data illuminate that methylcytosine experiences strong non-covalent interactions with the aerolysin nanopore at Region 1 around R220 than cytosine, which produces characteristic frequency-energy spectra. Furthermore, we experimentally validate the obtained hypothesis from frequency-energy spectra by designing single-site mutation of K238G which creates significantly enhanced non-covalent interactions for the recognition of methylcytosine. The frequency-energy spectrum of ions flowing inside membrane channels constitutes a single-molecule interaction spectrum, which bridges the gap between traditional ionic current recording and the MD simulations, facilitating the qualitative and quantitive description of the non-covalent interactions inside membrane channels.

scholarly journals Dynamic Single-molecule Colocalization Imaging - A New Method For Examining Membrane Protein Association In Living Cells

2009 ◽  
Vol 96 (3) ◽  
pp. 25a
Author(s):  
James T. McColl ◽  
Ricardo Alexandre ◽  
John R. James ◽  
Paul D. Dunne ◽  
Ji Won Yoon ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Single Molecule ◽  
Living Cells ◽  
New Method ◽  
Protein Association

scholarly journals A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein

2007 ◽  
Vol 104 (18) ◽  
pp. 7682-7687 ◽  
Author(s):  
Matthew R. Whorton ◽  
Michael P. Bokoch ◽  
Søren G. F. Rasmussen ◽  
Bo Huang ◽  
Richard N. Zare ◽  
...  
Keyword(s):  
High Density Lipoprotein ◽  
G Protein ◽  
G Proteins ◽  
Single Molecule ◽  
Density Lipoprotein ◽  
High Density ◽  
Phospholipid Bilayer ◽  
X Ray Crystallography ◽  
Signaling Complex ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) respond to a diverse array of ligands, mediating cellular responses to hormones and neurotransmitters, as well as the senses of smell and taste. The structures of the GPCR rhodopsin and several G proteins have been determined by x-ray crystallography, yet the organization of the signaling complex between GPCRs and G proteins is poorly understood. The observations that some GPCRs are obligate heterodimers, and that many GPCRs form both homo- and heterodimers, has led to speculation that GPCR dimers may be required for efficient activation of G proteins. However, technical limitations have precluded a definitive analysis of G protein coupling to monomeric GPCRs in a biochemically defined and membrane-bound system. Here we demonstrate that a prototypical GPCR, the β2-adrenergic receptor (β2AR), can be incorporated into a reconstituted high-density lipoprotein (rHDL) phospholipid bilayer particle together with the stimulatory heterotrimeric G protein, Gs. Single-molecule fluorescence imaging and FRET analysis demonstrate that a single β2AR is incorporated per rHDL particle. The monomeric β2AR efficiently activates Gs and displays GTP-sensitive allosteric ligand-binding properties. These data suggest that a monomeric receptor in a lipid bilayer is the minimal functional unit necessary for signaling, and that the cooperativity of agonist binding is due to G protein association with a receptor monomer and not receptor oligomerization.

scholarly journals Magnetically Oriented Phospholipid Bilayer Discs for Membrane Protein NMR

2018 ◽  
Vol 114 (3) ◽  
pp. 22a-23a
Author(s):  
Sang Ho Park ◽  
Jasmina Radoicic ◽  
Stanley J. Opella
Keyword(s):  
Membrane Protein ◽  
Protein Nmr ◽  
Phospholipid Bilayer

scholarly journals Cost-efficient nanoscopy reveals nanoscale architecture of liver cells and platelets

Nanophotonics ◽  
2019 ◽  
Vol 8 (7) ◽  
pp. 1299-1313 ◽  
Author(s):  
Hong Mao ◽  
Robin Diekmann ◽  
Hai Po H. Liang ◽  
Victoria C. Cogger ◽  
David G. Le Couteur ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Endothelial Cells ◽  
Single Molecule ◽  
Nanometer Scale ◽  
Color Channel ◽  
Liver Sinusoidal Endothelial Cells ◽  
Localization Microscopy ◽  
Cost Efficient ◽  
Core Facilities ◽  
Single Molecule Localization Microscopy

AbstractSingle-molecule localization microscopy (SMLM) provides a powerful toolkit to specifically resolve intracellular structures on the nanometer scale, even approaching resolution classically reserved for electron microscopy (EM). Although instruments for SMLM are technically simple to implement, researchers tend to stick to commercial microscopes for SMLM implementations. Here we report the construction and use of a “custom-built” multi-color channel SMLM system to study liver sinusoidal endothelial cells (LSECs) and platelets, which costs significantly less than a commercial system. This microscope allows the introduction of highly affordable and low-maintenance SMLM hardware and methods to laboratories that, for example, lack access to core facilities housing high-end commercial microscopes for SMLM and EM. Using our custom-built microscope and freely available software from image acquisition to analysis, we image LSECs and platelets with lateral resolution down to about 50 nm. Furthermore, we use this microscope to examine the effect of drugs and toxins on cellular morphology.

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