Single Molecule Approaches in RNA-Protein Interactions

2016 ◽  
pp. 89-106
Author(s):  
Victor Serebrov ◽  
Melissa J. Moore
Keyword(s):  
Single Molecule ◽  
Protein Interactions

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 pull-down for studying protein interactions

2012 ◽  
Vol 7 (3) ◽  
pp. 445-452 ◽  
Author(s):  
Ankur Jain ◽  
Ruijie Liu ◽  
Yang K Xiang ◽  
Taekjip Ha
Keyword(s):  
Single Molecule ◽  
Protein Interactions

scholarly journals mRNA structural dynamics shape Argonaute-target interactions

2019 ◽  
Author(s):  
Suzan Ruijtenberg ◽  
Stijn Sonneveld ◽  
Tao Ju Cui ◽  
Ive Logister ◽  
Dion de Steenwinkel ◽  
...  
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
Protein Interactions ◽  
Small Rnas ◽  
Mrna Degradation ◽  
Translational Repression ◽  
Modeling And Experiments ◽  
Regulate Protein ◽  
Sirna Target

AbstractSmall RNAs (such as miRNAs, siRNAs and piRNAs) regulate protein expression in a wide variety of biological processes and play an important role in cellular function, development and disease. Association of small RNAs with Argonaute (AGO) family proteins guide AGO to target RNAs, generally resulting in target silencing through transcriptional silencing, translational repression or mRNA degradation. Here we develop a live-cell single-molecule imaging assay to simultaneously visualize translation of individual mRNA molecules and their silencing by human AGO2-siRNA complexes. We find that siRNA target sites are commonly masked in vivo by RNA secondary structures, which inhibit AGO2-target interactions. Translating ribosomes unmask AGO2 binding sites, stimulating AGO2-target interactions and promoting mRNA degradation. Using a combination of mathematical modeling and experiments, we find that mRNA structures are highly heterogeneous and continuously refolding. We show that structural dynamics of mRNAs shape AGO2-target recognition, which may be a common feature controlling mRNA-protein interactions.

scholarly journals High-throughput single-molecule analysis of DNA–protein interactions by tethered particle motion

2012 ◽  
Vol 40 (12) ◽  
pp. e89-e89 ◽  
Author(s):  
Thomas Plénat ◽  
Catherine Tardin ◽  
Philippe Rousseau ◽  
Laurence Salomé
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Protein Interactions ◽  
Particle Motion ◽  
Tethered Particle Motion ◽  
Single Molecule Analysis ◽  
Dna Protein Interactions
Sign in / Sign up

Export Citation Format

Share Document