Halide and Proton Binding Kinetics of Yellow Fluorescent Protein Variants

Biochemistry ◽  
2013 ◽  
Vol 52 (14) ◽  
pp. 2482-2491 ◽  
Author(s):  
Harriet E. Seward ◽  
Jaswir Basran ◽  
Roanne Denton ◽  
Mark Pfuhl ◽  
Frederick W. Muskett ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Binding Kinetics ◽  
Proton Binding ◽  
Protein Variants ◽  
Kinetics Of

Kinetics of denaturation of the yellow fluorescent protein from coral zFP538

2005 ◽  
Author(s):  
Nadya N. Zubova ◽  
Vadim A. Korolenko ◽  
Alexander P. Savitsky
Keyword(s):  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Kinetics Of

scholarly journals Dual labeling of the fibronectin matrix and actin cytoskeleton with green fluorescent protein variants

2002 ◽  
Vol 115 (6) ◽  
pp. 1221-1229 ◽  
Author(s):  
Tomoo Ohashi ◽  
Daniel P. Kiehart ◽  
Harold P. Erickson
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filament ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Actin Binding ◽  
Matrix Assembly ◽  
Actin Binding Domain ◽  
Filament Bundles ◽  
Green Fluorescent ◽  
Protein Variants

We have prepared 3T3 cells doubly labeled to visualize simultaneously the extracellular fibronectin (FN) matrix and intracellular actin cytoskeleton in living cell cultures. We used FN-yellow fluorescent protein (FN-yfp) for the FN matrix, and the actin-binding domain of moesin fused to cyan fluorescent protein (cfp-Moe) to stain actin. Actin filament bundles were clearly seen in the protruding lamellae of the cells. FN matrix assembly appeared to be initiated as small spots of FN at the ends of actin filament bundles. The spots then elongated along the actin filament bundle toward the cell center to form FN fibrils. The end of the fibril towards the cell edge appeared immobile, and probably attached to the substrate, whereas the end toward the cell center frequently showed movements, suggesting attachment to the cell. Combining our data with the observations of Pankov et al. we suggest that fibrils grow by stretching this mobile end toward the cell center while adding new FN molecules at the end and along the entire lenght. When the cell culture was treated with cytochalasin to disrupt the actin cytoskeleton, some fibrils contracted substantially, suggesting that the segment attached primarily to the cell surface is stretched.

Insulin secretion by ‘kiss-and-run’ exocytosis in clonal pancreatic islet β-cells

2003 ◽  
Vol 31 (4) ◽  
pp. 833-836 ◽  
Author(s):  
T. Tsuboi ◽  
G.A. Rutter
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Secretory Vesicle ◽  
Β Cells ◽  
Vesicle Membrane ◽  
Synaptotagmin Iv ◽  
Green Fluorescent ◽  
Kinetics Of

Exocytotic release of neuropeptides and hormones is generally believed to involve the complete merger of the secretory vesicle with the plasma membrane. However, recent data have suggested that ‘kiss-and-run’ mechanisms may also play a role. To analyse secretory events in neuroendocrine β-cells, we imaged chimaeric reporters targeted to either the vesicle membrane [chimaeras of synaptobrevin-2 and pH-sensitive green fluorescent protein (synapto·pHluorin) or of phogrin (phosphatase on the granule of insulinoma) and enhanced green fluorescent protein (EGFP) (phogrin·EGFP)] or the lumen [neuropeptide Y (NPY)·pH-insensitive yellow fluorescent protein (Venus)] by evanescent wave microscopy. Unexpectedly, the frequency of NPY·Venus release events was only 17–27% of that of vesicle fusion reported with synapto·pHluorin, but not phogrin·EGFP, indicating that exocytosis of cargo peptides that is likely to require complete collapse of the vesicle into the plasma membrane is relatively rare. However, both the frequency and the kinetics of NPY·Venus release were modulated by stimulus strength or by overexpression of synaptotagmin IV, demonstrating the plasticity of ‘kiss-and-run’ fusion.

scholarly journals A machine-learning-guided mutagenesis platform for accelerated discovery of novel functional proteins

2018 ◽  
Author(s):  
Yutaka Saito ◽  
Misaki Oikawa ◽  
Hikaru Nakazawa ◽  
Teppei Niide ◽  
Tomoshi Kameda ◽  
...  
Keyword(s):  
Machine Learning ◽  
Molecular Evolution ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Screening Experiments ◽  
Novel Approach ◽  
Machine Learning Model ◽  
High Enrichment ◽  
Green Fluorescent ◽  
Protein Variants

AbstractMolecular evolution based on mutagenesis is widely used in protein engineering. However, optimal proteins are often difficult to obtain due to a large sequence space that requires high costs for screening experiments. Here, we propose a novel approach that combines molecular evolution with machine learning. In this approach, we conduct two rounds of mutagenesis where an initial library of protein variants is used to train a machine-learning model to guide mutagenesis for the second-round library. This enables to prepare a small library suited for screening experiments with high enrichment of functional proteins. We demonstrated a proof-of-concept of our approach by altering the reference green fluorescent protein (GFP) so that its fluorescence is changed to yellow while improving its fluorescence intensity. Using 155 and 78 variants for the initial and the second-round libraries, respectively, we successfully obtained a number of proteins showing yellow fluorescence, 12 of which had better fluorescence performance than the reference yellow fluorescent protein (YFP). These results show the potential of our approach as a powerful platform for accelerated discovery of functional proteins.

Binding kinetics of fluorescent bisphosphonates as a tool for monitoring bone dynamics in vivo

2013 ◽  
Author(s):  
Robert Tower ◽  
Graeme Campbell ◽  
Marc Muller ◽  
Olga Will ◽  
Frederieka Grundmann ◽  
...  
Keyword(s):  
Binding Kinetics ◽  
Kinetics Of ◽  
Bone Dynamics

Influence of Brain Gangliosides on the Formation and Properties of Supported Lipid Bilayers for Binding Kinetics Studies

2018 ◽  
Author(s):  
Luke Jordan ◽  
Nathan Wittenberg
Keyword(s):  
Lipid Bilayers ◽  
Binding Kinetics ◽  
Supported Lipid Bilayers ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Supported Lipid Bilayer ◽  
Head Groups ◽  
Lipid Diffusion ◽  
Kinetics Of ◽  
Brain Gangliosides

This is a comprehensive study of the effects of the four major brain gangliosides (GM1, GD1b, GD1a, and GT1b) on the adsorption and rupture of phospholipid vesicles on SiO2 surfaces for the formation of supported lipid bilayer (SLB) membranes. Using quartz crystal microbalance with dissipation monitoring (QCM-D) we show that gangliosides GD1a and GT1b significantly slow the SLB formation process, whereas GM1 and GD1b have smaller effects. This is likely due to the net ganglioside charge as well as the positions of acidic sugar groups on ganglioside glycan head groups. Data is included that shows calcium can accelerate the formation of ganglioside-rich SLBs. Using fluorescence recovery after photobleaching (FRAP) we also show that the presence of gangliosides significantly reduces lipid diffusion coefficients in SLBs in a concentration-dependent manner. Finally, using QCM-D and GD1a-rich SLB membranes we measure the binding kinetics of an anti-GD1a antibody that has similarities to a monoclonal antibody that is a hallmark of a variant of Guillain-Barre syndrome.

scholarly journals CO Binding Kinetics of Human Cytochrome P450 3A4

1995 ◽  
Vol 270 (10) ◽  
pp. 5014-5018 ◽  
Author(s):  
Aditya P. Koley ◽  
Jeroen T. M. Buters ◽  
Richard C. Robinson ◽  
Allen Markowitz ◽  
Fred K. Friedman
Keyword(s):  
Cytochrome P450 ◽  
Binding Kinetics ◽  
Cytochrome P450 3A4 ◽  
Human Cytochrome ◽  
Kinetics Of
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