Journal ArticleDOI
Mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity.
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TLDR
Improved biarsenical-tetracysteine motifs should enable detection of a much broader spectrum of cellular proteins, culminating in a >20-fold increase in contrast.Abstract:
Membrane-permeant biarsenical dyes such as FlAsH and ReAsH fluoresce upon binding to genetically encoded tetracysteine motifs expressed in living cells, yet spontaneous nonspecific background staining can prevent detection of weakly expressed or dilute proteins. If the affinity of the tetracysteine peptide could be increased, more stringent dithiol washes should increase the contrast between specific and nonspecific staining. Residues surrounding the tetracysteine motif were randomized and fused to GFP, retrovirally transduced into mammalian cells and iteratively sorted by fluorescence-activated cell sorting for high FRET from GFP to ReAsH in the presence of increasing concentrations of dithiol competitors. The selected sequences show higher fluorescence quantum yields and markedly improved dithiol resistance, culminating in a >20-fold increase in contrast. The selected tetracysteine sequences, HRWCCPGCCKTF and FLNCCPGCCMEP, maintain their enhanced properties as fusions to either terminus of GFP or directly to beta-actin. These improved biarsenical-tetracysteine motifs should enable detection of a much broader spectrum of cellular proteins.read more
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Dissertation
Mapping the proteins of the herpes simplex virus type 1 capsid
TL;DR: The aims of the work presented in this thesis were to use a variety of mutagenesis techniques to investigate the proteins of the HSV-1 capsid, and to show that the first 45 amino acids of VP19C were not essential for assembly of functional capsids and infectious virus particles.
DissertationDOI
GDI-Mediated Cdc42 Recycling at Polar Cortex in Dynamic Maintenance of Cell Polarity in Saccharomyces cerevisiae
TL;DR: This in depth study revealed that CDC42 at yeast polar cortex is dynamically maintained via two redundant pathways of distinct response time, and demonstrated Cdc42 GTPase cycle as the common regulator of actin and Rdil mediated pathways, a process supports their concentric localization.
Dissertation
Die Rolle von p6* für den HI-viralen Replikationszyklus
TL;DR: The p6*-carboxylterminus of the transframe-Protein (p6*)-CARB-CARB as mentioned in this paper is the bestandteil of the Gag-Pol-Polyproteinvorlaufers.
Book ChapterDOI
Mobility and Signaling of Single Receptor Proteins
Michael Prummer,Horst Vogel +1 more
Green fluorescent protein based indicators of dynamic redox changes and reactive oxygen species
TL;DR: The redox probes under physiological redox changes during superoxide bursts in macrophage cells, hyperoxic and hypoxic conditions, and in responses to H2O2-stimulating agents, e.g. epidermal growth factor and lysophosphatidic acid are investigated.
References
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The green fluorescent protein
TL;DR: In just three years, the green fluorescent protein from the jellyfish Aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochemistry and cell biology.
PatentDOI
FACS-optimized mutants of the green fluorescent protein (GFP)
TL;DR: In this article, three classes of GFP mutants having single excitation maxima around 488 nm are shown to be brighter than wild-type GFP following 488-nm excitation.
PatentDOI
Green fluorescent protein
Martin Chalfie,Douglas Prasher +1 more
TL;DR: This demonstration indicated that GFP could be used as a marker of gene expression and protein localization in living and fixed tissues and variations with more intense fluorescence or alterations in the excitation and emission spectra have been produced.
Journal ArticleDOI
Specific Covalent Labeling of Recombinant Protein Molecules Inside Live Cells
TL;DR: This system provides a recipe for slightly modifying a target protein so that it can be singled out from the many other proteins inside live cells and fluorescently stained by small nonfluorescent dye molecules added from outside the cells.
Journal ArticleDOI
An improved cyan fluorescent protein variant useful for FRET.
TL;DR: Cerulean is 2.5-fold brighter than ECFP and replacement of ECFP with Cerulean substantially improves the signal-to-noise ratio of a FRET-based sensor for glucokinase activation.