scholarly journals Optimized green fluorescent protein fused to FoF1-ATP synthase for single-molecule FRET using a fast anti-Brownian electrokinetic trap

2016 ◽  
Author(s):  
Maria Dienerowitz ◽  
Mykhailo Ilchenko ◽  
Bertram Su ◽  
Gabriele Deckers-Hebestreit ◽  
Günter Mayer ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Atp Synthase ◽  
Single Molecule ◽  
Fluorescent Protein ◽  
Single Molecule Fret ◽  
Green Fluorescent ◽  
Fof1 Atp Synthase

Single-Molecule Microscopy of the Green Fluorescent Protein Using Simultaneous Two-Color Excitation

ChemPhysChem ◽  
2001 ◽  
Vol 2 (6) ◽  
pp. 392-396 ◽  
Author(s):  
Gregor Jung ◽  
Jens Wiehler ◽  
Boris Steipe ◽  
Christoph Bräuchle ◽  
Andreas Zumbusch
Keyword(s):  
Green Fluorescent Protein ◽  
Single Molecule ◽  
Fluorescent Protein ◽  
Single Molecule Microscopy ◽  
Green Fluorescent

scholarly journals Single-Molecule Imaging and Computational Microscopy Approaches Clarify the Mechanism of the Dimerization and Membrane Interactions of Green Fluorescent Protein

2019 ◽  
Vol 20 (6) ◽  
pp. 1410 ◽  
Author(s):  
Xiaohua Wang ◽  
Kai Song ◽  
Yang Li ◽  
Ling Tang ◽  
Xin Deng
Keyword(s):  
Molecular Dynamics ◽  
Green Fluorescent Protein ◽  
Single Molecule ◽  
Fluorescent Protein ◽  
Hydrophobic Interactions ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Single Mutation ◽  
Functional Protein ◽  
Green Fluorescent

Green fluorescent protein (GFP) is widely used as a biomarker in living systems; however, GFP and its variants are prone to forming low-affinity dimers under physiological conditions. This undesirable tendency is exacerbated when fluorescent proteins (FP) are confined to membranes, fused to naturally-oligomeric proteins, or expressed at high levels in cells. Oligomerization of FPs introduces artifacts into the measurement of subunit stoichiometry, as well as interactions between proteins fused to FPs. Introduction of a single mutation, A206K, has been shown to disrupt hydrophobic interactions in the region responsible for GFP dimerization, thereby contributing to its monomerization. Nevertheless, a detailed understanding of how this single amino acid-dependent inhibition of dimerization in GFP occurs at the atomic level is still lacking. Single-molecule experiments combined with computational microscopy (atomistic molecular dynamics) revealed that the amino group of A206 contributes to GFP dimer formation via a multivalent electrostatic interaction. We further showed that myristoyl modification is an efficient mechanism to promote membrane attachment of GFP. Molecular dynamics-based site-directed mutagenesis has been used to identify the key functional residues in FPs. The data presented here have been utilized as a monomeric control in downstream single-molecule studies, facilitating more accurate stoichiometry quantification of functional protein complexes in living cells.

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