scholarly journals Mechanical unfold and transport of Green Florescent Protein through a nanopore

2018 ◽  
Author(s):  
Muhammad Adnan Shahzad
Keyword(s):  
Topological Structure ◽  
Fluorescent Protein ◽  
Alpha Helix ◽  
Coarse Grained ◽  
Constant Force ◽  
Go Model ◽  
Green Florescent Protein ◽  
Green Fluorescent ◽  
Loop Conformation ◽  
Native Fold

AbstractWe report the unfold and trans-location of Green Fluorescent protein (GFP) mechanically by a constant force acting parallel along the axis of nanopore. A coarse-grained numerical model (Go-model) were implemented both for the protein and the nanopore. Detail description of each peptide unfold by the constant force is presented. Depending on the GFP topological structure, β-sheet barrel, the protein unfold and transport as a double loop conformation in the confinement geometry. The result is compared with maltose binding protein (MBP), having majority of alpha helix, which unfold and trans-locate as single profile conformation through nanopore. The result emphasis that protein with different topological structure unfold and trans-locate in different fashion depending on their native fold structure.

scholarly journals GFP fusion protein with embedded foreign peptide

2019 ◽  
Author(s):  
K.A. Glukhova ◽  
V.G. Klyashtorny ◽  
B.S. Melnik
Keyword(s):  
Green Fluorescent Protein ◽  
Tertiary Structure ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Chimeric Protein ◽  
Bound Water ◽  
Alpha Helix ◽  
Point Of View ◽  
Foreign Peptide ◽  
Green Fluorescent

AbstractFrom the point of view structural biology and protein engineering the green fluorescent protein (GFP) is an exceptionally attracting object. The tertiary structure of GFP is quite unique: it reminds a “cylinder” or a “barrel” consisting of beta-layers that contains an alpha-helix inside. The “barrel” is a special container for an alpha-helix serving to protect the latter from the influence of the surroundings. Therefore a reasonable question arises whether the “barrel” can function as a container for preservation and isolation of other peptides. The alpha-helix itself contains hydrophilic amino acids, whereas inside the barrel there are many molecules of bound water. We supposed that the central alpha-helix of green fluorescent protein could be substituted for foreign peptide. In this study we checked the possibility for creation of such a system on base of GFP, where the toxic peptide is isolated from the environment inside the protein. The modification of green fluorescent protein was carried out. An antimicrobial peptide was inserted into the central alpha-helix. The results of our experiments show that such a chimeric protein is compact, soluble and non-toxic for the producing cell culture, but its structure is destabilized. The obtained data show that the idea of use of green fluorescent proteins as a «container» for storing foreign peptides could be realized.

scholarly journals The Potential for Green Fluorescent Protein as a Screening Tool in the Production of Haploid Potato

HortScience ◽  
2004 ◽  
Vol 39 (4) ◽  
pp. 775B-775
Author(s):  
Rose E. Palumbo ◽  
Richard E. Veilleux*
Keyword(s):  
Fluorescent Protein ◽  
Leaf Disc ◽  
Recessive Gene ◽  
Kanamycin Resistance ◽  
Leaf Discs ◽  
Diploid Clone ◽  
Green Florescent Protein ◽  
Green Fluorescent ◽  
Double Copy ◽  
Curled Leaves

A hybrid between a highly regenerative diploid clone (BARD 1-3) of Solanum phureja and haploid inducer IVP 101 was transformed with Agrobacterium tumefaciens strain 4404 containing plasmid pHB2892 with genes for green florescent protein (GFP) and kanamycin resistance. Hemizygous primary transformants (To) were produced from three leaf discs: 17 diploid plants from one leaf disc, three and nine tetraploids from the other two leaf discs. GFP expression was observed qualitatively under fluorescence microscopes and quantitatively with a GFP meter. Segregation ratios for tetraploid T1 seedlings fit models for single duplex insertions (35 transgenic: 1 non) or double simplex insertions (15 transgenic: 1 non). Diploid T1 seedlings segregated for deleterious traits: dwarfed size and curled leaves, as well as the GFP transgene. Similar segregation patterns in diploid families implied that all diploids may have been from the same transformation event. The cumulative segregation showed the dwarfed and curled plants fit a single recessive gene ratio (3 normal: 1 mutant), and GFP fit a double-copy insertion ratio (15 transgenic: 1 non). Six T1 selections were free of deleterious traits, consistently high expressers of GFP, and produced fertile pollen.

scholarly journals Mutational Analysis of Fibrillarin and Its Mobility in Living Human Cells

2000 ◽  
Vol 151 (3) ◽  
pp. 653-662 ◽  
Author(s):  
Sabine Snaar ◽  
Karien Wiesmeijer ◽  
Aart G. Jochemsen ◽  
Hans J. Tanke ◽  
Roeland W. Dirks
Keyword(s):  
Fluorescent Protein ◽  
Mutational Analysis ◽  
Diffusion Constant ◽  
Alpha Helix ◽  
Time Lapse ◽  
Human Cells ◽  
Low Frequencies ◽  
Mobile Fraction ◽  
Green Fluorescent ◽  
Carboxy Terminal

Cajal bodies (CBs) are subnuclear organelles that contain components of a number of distinct pathways in RNA transcription and RNA processing. CBs have been linked to other subnuclear organelles such as nucleoli, but the reason for the presence of nucleolar proteins such as fibrillarin in CBs remains uncertain. Here, we use full-length fibrillarin and truncated fibrillarin mutants fused to green fluorescent protein (GFP) to demonstrate that specific structural domains of fibrillarin are required for correct intranuclear localization of fibrillarin to nucleoli and CBs. The second spacer domain and carboxy terminal alpha-helix domain in particular appear to target fibrillarin, respectively, to the nucleolar transcription centers and CBs. The presence of the RNP domain seems to be a prerequisite for correct targeting of fibrillarin. Time-lapse confocal microscopy of human cells that stably express fibrillarin-GFP shows that CBs fuse and split, albeit at low frequencies. Recovered fluorescence of fibrillarin-GFP in nucleoli and CBs after photobleaching indicates that it is highly mobile in both organelles (estimated diffusion constant ∼0.02 μm2 s−1), and has a significantly larger mobile fraction in CBs than in nucleoli.

scholarly journals The Transmembrane Helix of the Escherichia coli Division Protein FtsI Localizes to the Septal Ring

2005 ◽  
Vol 187 (1) ◽  
pp. 320-328 ◽  
Author(s):  
Mark C. Wissel ◽  
Jennifer L. Wendt ◽  
Calista J. Mitchell ◽  
David S. Weiss
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Transmembrane Helix ◽  
Alpha Helix ◽  
Alanine Scanning Mutagenesis ◽  
Amino Terminal ◽  
Acid Fragment ◽  
Green Fluorescent ◽  
Septal Localization ◽  
A Site

ABSTRACT FtsI (also called PBP3) of Escherichia coli is a transpeptidase required for synthesis of peptidoglycan in the division septum and is one of about a dozen division proteins that localize to the septal ring. FtsI comprises a short amino-terminal cytoplasmic domain, a single transmembrane helix (TMH), and a large periplasmic domain that encodes the catalytic (transpeptidase) activity. We show here that a 26-amino-acid fragment of FtsI is sufficient to direct green fluorescent protein to the septal ring in cells depleted of wild-type FtsI. This fragment extends from W22 to V47 and corresponds to the TMH. This is a remarkable finding because it is usual for a TMH to target a protein to a site more specific than the membrane. Alanine-scanning mutagenesis of the TMH identified several residues important for septal localization. These residues cluster on one side of an alpha-helix, which we propose interacts directly with another division protein to recruit FtsI to the septal ring.

A Unified Model for Photophysical and Electro-Optical Properties of Green Fluorescent Proteins

2019 ◽  
Author(s):  
Chi-Yun Lin ◽  
Matthew Romei ◽  
Luke Oltrogge ◽  
Irimpan Mathews ◽  
Steven Boxer
Keyword(s):  
Driving Force ◽  
Fluorescent Protein ◽  
Charge Transfer Band ◽  
Photophysical Properties ◽  
Polymethine Dyes ◽  
Emission Rates ◽  
Unified Framework ◽  
Underlying Factor ◽  
Green Fluorescent ◽  
Protein Environment

Green fluorescent protein (GFPs) have become indispensable imaging and optogenetic tools. Their absorption and emission properties can be optimized for specific applications. Currently, no unified framework exists to comprehensively describe these photophysical properties, namely the absorption maxima, emission maxima, Stokes shifts, vibronic progressions, extinction coefficients, Stark tuning rates, and spontaneous emission rates, especially one that includes the effects of the protein environment. In this work, we study the correlations among these properties from systematically tuned GFP environmental mutants and chromophore variants. Correlation plots reveal monotonic trends, suggesting all these properties are governed by one underlying factor dependent on the chromophore's environment. By treating the anionic GFP chromophore as a mixed-valence compound existing as a superposition of two resonance forms, we argue that this underlying factor is defined as the difference in energy between the two forms, or the driving force, which is tuned by the environment. We then introduce a Marcus-Hush model with the bond length alternation vibrational mode, treating the GFP absorption band as an intervalence charge transfer band. This model explains all the observed strong correlations among photophysical properties; related subtopics are extensively discussed in Supporting Information. Finally, we demonstrate the model's predictive power by utilizing the additivity of the driving force. The model described here elucidates the role of the protein environment in modulating photophysical properties of the chromophore, providing insights and limitations for designing new GFPs with desired phenotypes. We argue this model should also be generally applicable to both biological and non-biological polymethine dyes.<br>

Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

2019 ◽  
Author(s):  
Jeffrey Chang ◽  
Matthew Romei ◽  
Steven Boxer
Keyword(s):  
Rational Design ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Unit Cell Size ◽  
Chlorine Substituent ◽  
Gfp Chromophore ◽  
The One ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

<p>Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of <i>cis</i> and <i>trans</i> rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the <i>trans</i> state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas in a tighter packing (7% smaller unit cell size), the hula-twist occurs.</p><p> </p><p> </p><p> </p><p> </p><p> </p><p> </p> <p> </p>

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