Reconstitution of Membrane Proteins into Polymer-Supported Membranes for Probing Diffusion and Interactions by Single Molecule Techniques

2011 ◽  
Vol 83 (17) ◽  
pp. 6792-6799 ◽  
Author(s):  
Friedrich Roder ◽  
Sharon Waichman ◽  
Dirk Paterok ◽  
Robin Schubert ◽  
Christian Richter ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Single Molecule ◽  
Supported Membranes ◽  
Polymer Supported

scholarly journals Polymer-Supported Membranes for Probing Transmembrane Protein Diffusion and Interaction by Single-Molecule Techniques

2011 ◽  
Vol 100 (3) ◽  
pp. 257a
Author(s):  
Friedrich Roder ◽  
Dirk Paterok ◽  
Sharon Waichman ◽  
Oliver Beutel ◽  
Jacob Piehler
Keyword(s):  
Single Molecule ◽  
Transmembrane Protein ◽  
Protein Diffusion ◽  
Supported Membranes ◽  
Polymer Supported

Rapid Transfer of Transmembrane Proteins for Single Molecule Dimerization Assays in Polymer-Supported Membranes

2014 ◽  
Vol 9 (11) ◽  
pp. 2479-2484 ◽  
Author(s):  
Friedrich Roder ◽  
Stephan Wilmes ◽  
Christian P. Richter ◽  
Jacob Piehler
Keyword(s):  
Single Molecule ◽  
Transmembrane Proteins ◽  
Supported Membranes ◽  
Polymer Supported ◽  
Rapid Transfer

scholarly journals Fast Diffusion of the Unassembled PetC1-GFP Protein in the Cyanobacterial Thylakoid Membrane

Life ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 15
Author(s):  
Radek Kaňa ◽  
Gábor Steinbach ◽  
Roman Sobotka ◽  
György Vámosi ◽  
Josef Komenda
Keyword(s):  
Membrane Proteins ◽  
Single Molecule ◽  
Protein Interactions ◽  
Thylakoid Membrane ◽  
Protein Complexes ◽  
Correlation Spectroscopy ◽  
Membrane Microdomains ◽  
Fast Diffusion ◽  
Light Harvesting Complexes ◽  
Term Survival

Biological membranes were originally described as a fluid mosaic with uniform distribution of proteins and lipids. Later, heterogeneous membrane areas were found in many membrane systems including cyanobacterial thylakoids. In fact, cyanobacterial pigment–protein complexes (photosystems, phycobilisomes) form a heterogeneous mosaic of thylakoid membrane microdomains (MDs) restricting protein mobility. The trafficking of membrane proteins is one of the key factors for long-term survival under stress conditions, for instance during exposure to photoinhibitory light conditions. However, the mobility of unbound ‘free’ proteins in thylakoid membrane is poorly characterized. In this work, we assessed the maximal diffusional ability of a small, unbound thylakoid membrane protein by semi-single molecule FCS (fluorescence correlation spectroscopy) method in the cyanobacterium Synechocystis sp. PCC6803. We utilized a GFP-tagged variant of the cytochrome b6f subunit PetC1 (PetC1-GFP), which was not assembled in the b6f complex due to the presence of the tag. Subsequent FCS measurements have identified a very fast diffusion of the PetC1-GFP protein in the thylakoid membrane (D = 0.14 − 2.95 µm2s−1). This means that the mobility of PetC1-GFP was comparable with that of free lipids and was 50–500 times higher in comparison to the mobility of proteins (e.g., IsiA, LHCII—light-harvesting complexes of PSII) naturally associated with larger thylakoid membrane complexes like photosystems. Our results thus demonstrate the ability of free thylakoid-membrane proteins to move very fast, revealing the crucial role of protein–protein interactions in the mobility restrictions for large thylakoid protein complexes.

scholarly journals Single Molecule Localization Microscopy (SMLM) imaging of bacteria: A sample preparation guide

2020 ◽  
Author(s):  
Sachith D. Gunasinghe ◽  
Kirstin D. Elgass ◽  
Toby D. M. Bell ◽  
Trevor Lithgow
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Single Molecule ◽  
Spatial Organization ◽  
Outer Membrane Proteins ◽  
Fluorescent Microscopy ◽  
Super Resolution ◽  
Model Systems ◽  
Invaluable Tool ◽  
Optical Reconstruction

Abstract In recent years Super-resolution microscopy has become an invaluable tool to noninvasively interrogate the membrane architecture of bacteria to study the spatial organization of proteins associated with membranes, which in turn help us to understand how bacteria have evolved to exploit environmental niches. Model systems like Escherichia coli and Caulobacter cresentus have been used to study the spatiotemporal organization of membrane proteins. Like most gram-negative bacteria, the outer membrane of E.coli is populated with β-barrel proteins, which serve as selective channels where exchange of small molecules take place. Surface exposed domains in these channels provide means to fluorescently label and utilise them for fluorescent microscopy studies to investigate their spatial organization at the outer membrane. Here, we describe a methodology to fluorescently label outer membrane proteins in E.coli and study their spatial organization using direct stochastic optical reconstruction microscopy (dSTORM).

scholarly journals Screening for Influx through Membrane Proteins on the Single-Molecule Level using an Automated and Parallel Lipid Bilayer Platform

2014 ◽  
Vol 106 (2) ◽  
pp. 559a
Author(s):  
Mohamed Kreir ◽  
Matthias Beckler ◽  
Astrid Seifert ◽  
Conrad Weichbrodt ◽  
Gerhard Baaken ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Lipid Bilayer ◽  
Single Molecule ◽  
Single Molecule Level

scholarly journals Single molecule fluorescence for membrane proteins

Methods ◽  
2018 ◽  
Vol 147 ◽  
pp. 221-228 ◽  
Author(s):  
Oliver K. Castell ◽  
Patricia M. Dijkman ◽  
Daniel N. Wiseman ◽  
Alan D. Goddard
Keyword(s):  
Membrane Proteins ◽  
Single Molecule ◽  
Single Molecule Fluorescence

scholarly journals Direct Detection of Intramolecular Dynamics of Membrane Proteins using Time-resolved X-ray Single-molecule Tracking

2020 ◽  
Vol 118 (3) ◽  
pp. 170a
Author(s):  
Kazuhiro Mio ◽  
shoko fujimura ◽  
Masaki Ishihara ◽  
Muneyo Mio ◽  
Masahiro Kuramochi ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Single Molecule ◽  
Direct Detection ◽  
Intramolecular Dynamics ◽  
Time Resolved ◽  
Single Molecule Tracking ◽  
X Ray

scholarly journals Synthetic and genetic dimers as quantification ruler for single-molecule counting with PALM

2019 ◽  
Vol 30 (12) ◽  
pp. 1369-1376 ◽  
Author(s):  
Tim N. Baldering ◽  
Marina S. Dietz ◽  
Karl Gatterdam ◽  
Christos Karathanasis ◽  
Ralph Wieneke ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Single Molecule ◽  
Fusion Proteins ◽  
Fluorescent Proteins ◽  
Superresolution Microscopy ◽  
Superresolution Imaging ◽  
Molecular Information ◽  
Kinetics Of

How membrane proteins oligomerize determines their function. Superresolution microscopy can report on protein clustering and extract quantitative molecular information. Here, we evaluate the blinking kinetics of four photoactivatable fluorescent proteins for quantitative single-molecule microscopy. We identified mEos3.2 and mMaple3 to be suitable for molecular quantification through blinking histogram analysis. We designed synthetic and genetic dimers of mEos3.2 as well as fusion proteins of monomeric and dimeric membrane proteins as reference structures, and we demonstrate their versatile use for quantitative superresolution imaging in vitro and in situ. We further found that the blinking behavior of mEos3.2 and mMaple3 is modified by a reducing agent, offering the possibility to adjust blinking parameters according to experimental needs.

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