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.

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 Oligomerization of green fluorescent protein in the secretory pathway of endocrine cells

2001 ◽  
Vol 360 (3) ◽  
pp. 645-649 ◽  
Author(s):  
Renu K. JAIN ◽  
Paul B. M. JOYCE ◽  
Miguel MOLINETE ◽  
Philippe A. HALBAN ◽  
Sven-Ulrik GORR
Keyword(s):  
Green Fluorescent Protein ◽  
Endocrine Cells ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Reporter Protein ◽  
Regulated Secretory Pathway ◽  
Green Fluorescent

Green fluorescent protein (GFP) is used extensively as a reporter protein to monitor cellular processes, including intracellular protein trafficking and secretion. In general, this approach depends on GFP acting as a passive reporter protein. However, it was recently noted that GFP oligomerizes in the secretory pathway of endocrine cells. To characterize this oligomerization and its potential role in GFP transport, cytosolic and secretory forms of enhanced GFP (EGFP) were expressed in GH4C1 and AtT-20 endocrine cells. Biochemical analysis showed that cytosolic EGFP existed as a 27kDa monomer, whereas secretory forms of EGFP formed disulphide-linked oligomers. EGFP contains two cysteine residues (Cys49 and Cys71), which could play a role in this oligomerization. Site-directed mutagenesis of Cys49 and Cys71 showed that both cysteine residues were involved in disulphide interactions. Substitution of either cysteine residue resulted in a reduction or loss of oligomers, although dimers of the secretory form of EGFP remained. Mutation of these residues did not adversely affect the fluorescence of EGFP. EGFP oligomers were stored in secretory granules and secreted by the regulated secretory pathway in endocrine AtT-20 cells. Similarly, the dimeric mutant forms of EGFP were still secreted via the regulated secretory pathway, indicating that the higher-order oligomers were not necessary for sorting in AtT-20 cells. These results suggest that the oligomerization of EGFP must be considered when the protein is used as a reporter molecule in the secretory pathway.

scholarly journals Computational prediction of the tolerance to amino-acid deletion in green-fluorescent protein

2016 ◽  
Author(s):  
Eleisha L. Jackson ◽  
Stephanie J. Spielman ◽  
Claus O. Wilke
Keyword(s):  
Green Fluorescent Protein ◽  
Protein Structure ◽  
Amino Acid ◽  
Protein Design ◽  
Fluorescent Protein ◽  
Computational Prediction ◽  
Single Amino Acid ◽  
Dimensional Structure ◽  
Amino Acid Deletion ◽  
Green Fluorescent

AbstractProteins evolve through two primary mechanisms: substitution, where mutations alter a protein’s amino-acid sequence, and insertions and deletions (indels), where amino acids are either added to or removed from the sequence. Protein structure has been shown to influence the rate at which substitutions accumulate across sites in proteins, but whether structure similarly constrains the occurrence of indels has not been rigorously studied. Here, we investigate the extent to which structural properties known to covary with protein evolutionary rates might also predict protein tolerance to indels. Specifically, we analyze a publicly available dataset of single–amino-acid deletion mutations in enhanced green fluorescent protein (eGFP) to assess how well the functional effect of deletions can be predicted from protein structure. We find that weighted contact number (WCN), which measures how densely packed a residue is within the protein’s three-dimensional structure, provides the best single predictor for whether eGFP will tolerate a given deletion. We additionally find that using protein design to explicitly model deletions results in improved predictions of functional status when combined with other structural predictors. Our work suggests that structure plays fundamental role in constraining deletions at sites in proteins, and further that similar biophysical constraints influence both substitutions and deletions. This study therefore provides a solid foundation for future work to examine how protein structure influences tolerance of more complex indel events, such as insertions or large deletions.

scholarly journals A molecular dynamics model for glycosylphosphatidyl-inositol anchors: “flop down” or “lollipop”?

2018 ◽  
Vol 20 (46) ◽  
pp. 29314-29324
Author(s):  
Pallavi Banerjee ◽  
Marko Wehle ◽  
Reinhard Lipowsky ◽  
Mark Santer
Keyword(s):  
Molecular Dynamics ◽  
Green Fluorescent Protein ◽  
Computational Model ◽  
Fluorescent Protein ◽  
Dynamics Model ◽  
Green Fluorescent ◽  
Glycosylphosphatidyl Inositol

Computational model for GPI anchors tested in DMPC and POPC bilayers. The free anchor rarely occurs as an erected “lollipop-like” conformation, it rather “flops down” onto the bilayer surface. Yet an attached protein (here green fluorescent protein) exhibits extensive orientational flexibility due to the phospho-ethanolamine linker.

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