Heuristic Statistical Analysis of Fluorescence Fluctuation Data with Bright Spikes: Application to Ligand Binding to the Human Serotonin Receptor Expressed in Escherichia coli Cells

2001 ◽  
Vol 382 (3) ◽  
pp. 355-361 ◽  
Author(s):  
Elke Van Craenenbroeck ◽  
Jo Vercammen ◽  
Gunther Matthys ◽  
Jan Beirlant ◽  
Christophe Marot ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ligand Binding ◽  
Serotonin Receptor ◽  
Specific Binding ◽  
Statistical Tests ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Escherichia Coli Cells ◽  
Multiple Binding Sites ◽  
Multiple Binding

Abstract A statistical method for the analysis of fluorescence fluctuation amplitudes including bright spikes is presented. This situation arises e. g. when fluorescent ligands interact with receptors carrying multiple binding sites. The technique gives information on the amount of bound ligand in solution, making it a complementary technique to fluorescence correlation spectroscopy analysis, which cannot be applied in this situation. Two simple statistical tests are proposed that can discriminate between fluorescence intensities originating from free ligands or complexes. The performance of the two tests is evaluated and compared on mixtures of a fluorophore and fluorophore coated beads that mimic the behaviour of multiliganded complexes. An application to ligand binding to the serotonin receptor, expressed on Escherichia coli cells, is also provided. Specific binding of a fluorophore to this receptor, as well as competition with several ligands, is assessed.

A fluorescence correlation spectroscopy study of ligand interaction with cytokinin-specific binding protein from mung bean

2010 ◽  
Vol 391 (1) ◽  
Author(s):  
Paweł Zawadzki ◽  
Genowefa Ślósarek ◽  
Jerzy Boryski ◽  
Przemysław Wojtaszek
Keyword(s):  
Ligand Binding ◽  
Diffusion Coefficients ◽  
Fluorescence Correlation Spectroscopy ◽  
Mung Bean ◽  
Specific Binding ◽  
Binding Protein ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation ◽  
Specific Binding Protein ◽  
Two Populations

Abstract Cytokinins are essential plant hormones that regulate numerous physiological processes. Recently, a protein was identified in mung bean (Vigna radiata) and characterized as a cytokinin-specific binding protein (VrCSBP). Fluorescence correlation spectroscopy was used to investigate the interaction between VrCSBP and its ligands. The synthetic cytokinin, N-phenyl-N′-(4-pyridyl) urea, was labeled with two fluorophores, 7-nitro-2,1,3-benzoxadiazole and rhodamine B. Protein-ligand binding was analyzed in an equilibrium saturation binding experiment and confirmed by the competition assay. Surprisingly, it was found that VrCSBP binds not only to cytokinins, but also to gibberellins. In addition, in the presence of natural cytokinins and gibberellins, two populations of VrCSBP that differ in their diffusion coefficients were detected. The diffusion coefficients of these two populations could be related to mono- and dimeric states, which suggests a new mode of operation in ligand binding by VrCSBP, in which dimerization induced by natural ligands enhances the ligand binding capacity of the protein.

scholarly journals Norovirus and Histo-Blood Group Antigens: Demonstration of a Wide Spectrum of Strain Specificities and Classification of Two Major Binding Groups among Multiple Binding Patterns

2005 ◽  
Vol 79 (11) ◽  
pp. 6714-6722 ◽  
Author(s):  
Pengwei Huang ◽  
Tibor Farkas ◽  
Weiming Zhong ◽  
Ming Tan ◽  
Scott Thornton ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Blood Group ◽  
Acute Gastroenteritis ◽  
Specific Binding ◽  
Wide Spectrum ◽  
Phylogenetic Analyses ◽  
Lewis Antigens ◽  
Blood Group Antigens ◽  
Multiple Binding

ABSTRACT Noroviruses, an important cause of acute gastroenteritis, have been found to recognize human histo-blood group antigens (HBGAs) as receptors. Four strain-specific binding patterns to HBGAs have been described in our previous report. In this study, we have extended the binding patterns to seven based on 14 noroviruses examined. The oligosaccharide-based assays revealed additional epitopes that were not detected by the saliva-based assays. The seven patterns have been classified into two groups according to their interactions with three major epitopes (A/B, H, and Lewis) of human HBGAs: the A/B-binding group and the Lewis-binding group. Strains in the A/B binding group recognize the A and/or B and H antigens, but not the Lewis antigens, while strains in the Lewis-binding group react only to the Lewis and/or H antigens. This classification also resulted in a model of the norovirus/HBGA interaction. Phylogenetic analyses showed that strains with identical or closely related binding patterns tend to be clustered, but strains in both binding group can be found in both genogroups I and II. Our results suggest that noroviruses have a wide spectrum of host range and that human HBGAs play an important role in norovirus evolution. The high polymorphism of the human HBGA system, the involvement of multiple epitopes, and the typical protein/carbohydrate interaction between norovirus VLPs and HBGAs provide an explanation for the virus-ligand binding diversities.

scholarly journals Fluorescence Correlation Spectroscopy in Drug Discovery: Study of Alexa532-Endothelin 1 Binding to the Endothelin ETAReceptor to Describe the Pharmacological Profile of Natural Products

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Catherina Caballero-George ◽  
Thomas Sorkalla ◽  
Daniel Jakobs ◽  
Jessica Bolaños ◽  
Huzefa Raja ◽  
...  
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Inositol Phosphate ◽  
Specific Binding ◽  
Correlation Spectroscopy ◽  
Ligand Complex ◽  
Receptor Ligand ◽  
Fluorescence Correlation ◽  
Fungal Extract ◽  
Dose Dependent Increase ◽  
Dose Dependent

Fluorescence correlation spectroscopy and the newly synthesized Alexa532-ET1 were used to study the dynamics of the endothelin ETAreceptor-ligand complex alone and under the influence of a semisynthetic selective antagonist and a fungal extract on living A10 cells. Dose-dependent increase of inositol phosphate production was seen for Alexa532-ET1, and its binding was reduced to 8% by the selective endothelin ETAantagonist BQ-123, confirming the specific binding of Alexa532-ET1 to the endothelin ETAreceptor. Two different lateral mobilities of the receptor-ligand complexes within the cell membrane were found allowing the discrimination of different states for this complex. BQ-123 showed a strong binding affinity to the “inactive” receptor state characterized by the slow diffusion time constant. A similar effect was observed for the fungal extract, which completely displaced Alexa532-ET1 from its binding to the “inactive” receptor state. These findings suggest that both BQ-123 and the fungal extract act as inverse agonists.

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