scholarly journals Peptide Signals Encode Protein Localization

2007 ◽  
Vol 189 (21) ◽  
pp. 7581-7585 ◽  
Author(s):  
Jay H. Russell ◽  
Kenneth C. Keiler
Keyword(s):  
Protein Interactions ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Protein Protein Interactions ◽  
Helical Structures ◽  
Bacterial Proteins ◽  
Complex Protein ◽  
Peptide Signals ◽  
Green Fluorescent ◽  
Localization Information

ABSTRACT Many bacterial proteins are localized to precise intracellular locations, but in most cases the mechanism for encoding localization information is not known. Screening libraries of peptides fused to green fluorescent protein identified sequences that directed the protein to helical structures or to midcell. These peptides indicate that protein localization can be encoded in 20-amino-acid peptides instead of complex protein-protein interactions and raise the possibility that the location of a protein within the cell could be predicted from bioinformatic data.

scholarly journals Application of a Chimeric Green Fluorescent Protein to Study Protein-Protein Interactions

BioTechniques ◽  
1997 ◽  
Vol 23 (5) ◽  
pp. 864-872 ◽  
Author(s):  
N. Garamszegi ◽  
Z.P. Garamszegi ◽  
M.S. Rogers ◽  
S.J. De-Marco ◽  
E.E. Strehler
Keyword(s):  
Green Fluorescent Protein ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Protein Protein Interactions ◽  
Green Fluorescent

Detecting Protein−Protein Interactions with a Green Fluorescent Protein Fragment Reassembly Trap:  Scope and Mechanism

2005 ◽  
Vol 127 (1) ◽  
pp. 146-157 ◽  
Author(s):  
Thomas J. Magliery ◽  
Christopher G. M. Wilson ◽  
Weilan Pan ◽  
Dennis Mishler ◽  
Indraneel Ghosh ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Protein Protein Interactions ◽  
Protein Fragment ◽  
Green Fluorescent

scholarly journals In Vivo Quantitative Estimation of DNA-Dependent Interaction of Sox2 and Oct4 Using BirA-Catalyzed Site-Specific Biotinylation

Biomolecules ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 142 ◽  
Author(s):  
Arman Kulyyassov ◽  
Vasily Ogryzko
Keyword(s):  
Transcription Factors ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Quantitative Estimation ◽  
Multiple Reaction Monitoring ◽  
Negative Control ◽  
Protein Protein Interactions ◽  
Close Proximity ◽  
Green Fluorescent

Protein–protein interactions of core pluripotency transcription factors play an important role during cell reprogramming. Cell identity is controlled by a trio of transcription factors: Sox2, Oct4, and Nanog. Thus, methods that help to quantify protein–protein interactions may be useful for understanding the mechanisms of pluripotency at the molecular level. Here, a detailed protocol for the detection and quantitative analysis of in vivo protein–protein proximity of Sox2 and Oct4 using the proximity-utilizing biotinylation (PUB) method is described. The method is based on the coexpression of two proteins of interest fused to a biotin acceptor peptide (BAP)in one case and a biotin ligase enzyme (BirA) in the other. The proximity between the two proteins leads to more efficient biotinylation of the BAP, which can be either detected by Western blotting or quantified using proteomics approaches, such as a multiple reaction monitoring (MRM) analysis. Coexpression of the fusion proteins BAP-X and BirA-Y revealed strong biotinylation of the target proteins when X and Y were, alternatively, the pluripotency transcription factors Sox2 and Oct4, compared with the negative control where X or Y was green fluorescent protein (GFP), which strongly suggests that Sox2 and Oct4 come in close proximity to each other and interact.

scholarly journals Intracellular Targeting of Gag Proteins of the Drosophila Telomeric Retrotransposons

2003 ◽  
Vol 77 (11) ◽  
pp. 6376-6384 ◽  
Author(s):  
S. Rashkova ◽  
A. Athanasiadis ◽  
M.-L. Pardue
Keyword(s):  
Protein Interactions ◽  
Fluorescent Protein ◽  
Cluster Formation ◽  
Intracellular Localization ◽  
Ltr Retrotransposons ◽  
Protein Protein Interactions ◽  
Gag Proteins ◽  
Intracellular Targeting ◽  
Identify Amino Acid ◽  
Green Fluorescent

ABSTRACT Drosophila has two non-long-terminal-repeat (non-LTR) retrotransposons that are unique because they have a defined role in chromosome maintenance. These elements, HeT-A and TART, extend chromosome ends by successive transpositions, producing long arrays of head-to-tail repeat sequences. These arrays appear to be analogous to the arrays produced by telomerase on chromosomes of other organisms. While other non-LTR retrotransposons transpose to many chromosomal sites, HeT-A and TART transpose only to chromosome ends. Although HeT-A and TART belong to different subfamilies of non-LTR retrotransposons, they encode very similar Gag proteins, which suggests that Gag proteins are involved in their unique transposition targeting. We have recently shown that both Gags localize efficiently to nuclei where HeT-A Gag forms structures associated with telomeres. TART Gag does not associate with telomeres unless HeT-A Gag is present, suggesting a symbiotic relationship in which HeT-A Gag provides telomeric targeting. We now report studies to identify amino acid regions responsible for different aspects of the intracellular targeting of these proteins. Green fluorescent protein-tagged deletion derivatives were expressed in cultured Drosophila cells. The intracellular localization of these proteins shows the following. (i) Several regions that direct subcellular localizations or cluster formation are found in both Gags and are located in equivalent regions of the two proteins. (ii) Regions important for telomere association are present only in HeT-A Gag. These are present at several places in the protein, are not redundant, and cannot be complemented in trans. (iii) Regions containing zinc knuckle and major homology region motifs, characteristic of retroviral Gags, are involved in protein-protein interactions of the telomeric Gags, as they are in retroviral Gags.

scholarly journals Strategies to improve the fluorescent signal of the tripartite sfGFP system

2020 ◽  
Vol 52 (9) ◽  
pp. 998-1006
Author(s):  
Jing Shen ◽  
Wenlu Zhang ◽  
Chunyang Gan ◽  
Xiafei Wei ◽  
Jie Li ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Bimolecular Fluorescence Complementation ◽  
Fluorescence Signal ◽  
Recovery Efficiency ◽  
Good Recovery ◽  
Protein Protein Interactions ◽  
Bifc Assay ◽  
Green Fluorescent

Abstract Bimolecular fluorescence complementation (BiFC) is a popular method used to detect protein–protein interactions. For a BiFC assay, a fluorescent protein is usually split into two parts, and the fluorescence is recovered upon the interaction between the fused proteins of interest. As an elegant extension of BiFC, a tripartite superfold green fluorescent protein (sfGFP) system that has the advantages of low background fluorescence and small fusion tag size has been developed. However, the tripartite system exhibits a low fluorescence signal in some cases. To address this problem, we proposed to increase the affinity between the two parts, G1–9 and G11, of the tripartite system by adding affinity pairs. Among the three affinity pairs tested, LgBiT-HiBiT improved both the signal and signal-to-noise (S/N) ratio to the greatest extent. More strikingly, the direct covalent fusion of G11 to G1–9, which converted the tripartite system into a new bipartite system, enhanced the S/N ratio from 20 to 146, which is superior to the bipartite sfGFP system split at 157/158 or 173/174. Our results implied that the 10th β-strand of sfGFP has a low affinity and a good recovery efficiency to construct a robust BiFC system, and this concept might be applied to other fluorescent proteins with similar structure to construct new BiFC systems.

Novel determinant of PKC-ε anchoring at cardiac Z-lines

2005 ◽  
Vol 289 (5) ◽  
pp. H1941-H1950 ◽  
Author(s):  
Seth L. Robia ◽  
Misuk Kang ◽  
Jeffery W. Walker
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Interactions ◽  
Cardiac Myocyte ◽  
Fluorescent Protein ◽  
Cardiac Cells ◽  
Protein Protein Interactions ◽  
Multiple Points ◽  
Myocyte Function ◽  
Green Fluorescent

The Z-line represents a critical link between the transverse tubule network and cytoskeleton of cardiac cells with a role in anchoring structural proteins, ion channels, and signaling molecules. Protein kinase C-ε (PKC-ε) regulates cardiac excitability, cardioprotection, and growth, possibly as a consequence of translocation to the Z-line/T tubule region. To investigate the mechanism of PKC-ε translocation, fragments of its NH2-terminal 144-amino acid variable domain, εV1, were fused with green fluorescent protein and evaluated by quantitative Fourier image analysis of decorated myocytes. Deletion of 23 amino acids from the NH2-terminus of εV1, including an EAVSLKPT motif important for binding to a receptor for activated C kinase (RACK2), reduced but did not abolish Z-line binding. Further deletions of up to 84 amino acids from the NH2-terminus of εV1 also did not prevent Z-line decoration. However, deletions of residues 85–144 from the COOH-terminus strongly reduced Z-line binding. COOH-terminal deletions caused 2.5-fold greater loss of binding energy (ΔΔG) than did NH2-terminal deletions. Synthetic peptides derived from these regions modulated εV1 binding and cardiac myocyte function, but also revealed considerable heterogeneity within populations of adult cardiac myocytes. The COOH-terminal subdomain important for Z-line anchoring maps to a surface in the εV1 crystal structure that complements the eight-amino acid RACK2 binding site and two previously identified membrane docking motifs. PKC-ε anchoring at the cardiac Z-line/T tubule appears to rely on multiple points of contact probably involving protein-lipid and protein-protein interactions.

Development and implementation of split-GFP-based bimolecular fluorescence complementation (BiFC) assays in yeast

2008 ◽  
Vol 36 (3) ◽  
pp. 479-482 ◽  
Author(s):  
Emma Barnard ◽  
Neil V. McFerran ◽  
Alan Trudgett ◽  
John Nelson ◽  
David J. Timson
Keyword(s):  
Protein Interactions ◽  
Fluorescent Protein ◽  
Model Organism ◽  
Subcellular Location ◽  
Bimolecular Fluorescence Complementation ◽  
Protein Protein Interactions ◽  
Yeast Saccharomyces Cerevisiae ◽  
Green Fluorescent ◽  
Fluorescence Complementation ◽  
Bimolecular Fluorescence

BiFC (bimolecular fluorescence complementation) is a tool for investigating interactions between proteins. Non-fluorescent fragments of, for example, GFP (green fluorescent protein) are fused to the interacting partners. The interaction brings the fragments together, which then fold, reassemble and fluoresce. This process can be carried out in living cells and provides information both on the interaction and its subcellular location. We have developed a split-GFP-based BiFC assay for use in the budding yeast Saccharomyces cerevisiae in which the modifications are carried out at the genomic level, thus resulting in the tagged yeast proteins being expressed at wild-type levels. The system is capable of detecting interactions in all subcellular compartments tested (the cytoplasm, mitochondria and nucleus) and makes a valuable addition to techniques for the investigation of protein–protein interactions in this model organism.

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