scholarly journals Studying Multi-Protein Interactions by Fluorescence Detected Sedimentation Velocity Combining Hydrodynamic Separation of Complexes with Fluorescence Quenching Analysis

2019 ◽  
Vol 116 (3) ◽  
pp. 194a
Author(s):  
Huaying Zhao ◽  
Siddhartha A. Datta ◽  
Sung Kim ◽  
Sumit K. Chaturvedi ◽  
Alan Rein ◽  
...  
Keyword(s):  
Fluorescence Quenching ◽  
Protein Interactions ◽  
Sedimentation Velocity ◽  
Quenching Analysis ◽  
Hydrodynamic Separation

The Inhibition of Polysialyltranseferase ST8SiaIV Through Heparin Binding to Polysialyltransferase Domain (PSTD)

2019 ◽  
Vol 15 (5) ◽  
pp. 486-495 ◽  
Author(s):  
Li-Xin Peng ◽  
Xue-Hui Liu ◽  
Bo Lu ◽  
Si-Ming Liao ◽  
Feng Zhou ◽  
...  
Keyword(s):  
Fluorescence Quenching ◽  
Polysialic Acid ◽  
Neuronal Cell ◽  
Cd Spectroscopy ◽  
Migration And Invasion ◽  
Heparin Binding ◽  
Clinical Prognosis ◽  
Quenching Analysis ◽  
Different Types ◽  
Α Helix

Background:The polysialic acid (polySia) is a unique carbohydrate polymer produced on the surface Of Neuronal Cell Adhesion Molecule (NCAM) in a number of cancer cells, and strongly correlates with the migration and invasion of tumor cells and with aggressive, metastatic disease and poor clinical prognosis in the clinic. Its synthesis is catalyzed by two polysialyltransferases (polySTs), ST8SiaIV (PST) and ST8SiaII (STX). Selective inhibition of polySTs, therefore, presents a therapeutic opportunity to inhibit tumor invasion and metastasis due to NCAM polysialylation. Heparin has been found to be effective in inhibiting the ST8Sia IV activity, but no clear molecular rationale. It has been found that polysialyltransferase domain (PSTD) in polyST plays a significant role in influencing polyST activity, and thus it is critical for NCAM polysialylation based on the previous studies.Objective:To determine whether the three different types of heparin (unfractionated hepain (UFH), low molecular heparin (LMWH) and heparin tetrasaccharide (DP4)) is bound to the PSTD; and if so, what are the critical residues of the PSTD for these binding complexes?Methods:Fluorescence quenching analysis, the Circular Dichroism (CD) spectroscopy, and NMR spectroscopy were used to determine and analyze interactions of PSTD-UFH, PSTD-LMWH, and PSTD-DP4.Results:The fluorescence quenching analysis indicates that the PSTD-UFH binding is the strongest and the PSTD-DP4 binding is the weakest among these three types of the binding; the CD spectra showed that mainly the PSTD-heparin interactions caused a reduction in signal intensity but not marked decrease in α-helix content; the NMR data of the PSTD-DP4 and the PSTDLMWH interactions showed that the different types of heparin shared 12 common binding sites at N247, V251, R252, T253, S257, R265, Y267, W268, L269, V273, I275, and K276, which were mainly distributed in the long α-helix of the PSTD and the short 3-residue loop of the C-terminal PSTD. In addition, three residues K246, K250 and A254 were bound to the LMWH, but not to DP4. This suggests that the PSTD-LMWH binding is stronger than the PSTD-DP4 binding, and the LMWH is a more effective inhibitor than DP4.Conclusion:The findings in the present study demonstrate that PSTD domain is a potential target of heparin and may provide new insights into the molecular rationale of heparin-inhibiting NCAM polysialylation.

A Fluorescence Quenching Analysis of the Binding of Fluoroquinolones to Humic Acid

2017 ◽  
Vol 71 (11) ◽  
pp. 2512-2518 ◽  
Author(s):  
Ryan P. Ferrie ◽  
Gregory E. Hewitt ◽  
Bruce D. Anderson
Keyword(s):  
Humic Acid ◽  
Fluorescence Quenching ◽  
Water Solubility ◽  
Binding Constants ◽  
High Water ◽  
Static Quenching ◽  
Temperature Range ◽  
Equilibrium Binding ◽  
Quenching Analysis ◽  
High Water Solubility

Fluorescence quenching was used to investigate the interaction of six fluoroquinolones with humic acid. Static quenching was observed for the binding of ciprofloxacin, enoxacin, fleroxacin, levofloxacin, norfloxacin, and ofloxacin to humic acid. The equilibrium binding constants were found from Stern–Volmer plots of the data. The quenching experiments were repeated over a temperature range of 25–45 ℃ and van’t Hoff plots were generated. From these linear plots, thermodynamic values were calculated for Δ H, Δ G, and Δ S for each of the fluoroquinolones. The equilibrium binding constants were found to be <1 for all the antibiotics studied. The calculated ΔH values were all negative and ranged from −9.5 to −27.6 kJ/mol. The high water solubility of the antibiotics and low ΔH of binding suggests that the antibiotics will be transported easily through the environment. Finally, whether the fluoroquinolones are in a protonated, deprotonated, or partially protonated state is found to correlate to the strength of binding to humic acid.

scholarly journals Investigation of 1-Methylcytosine as a Ligand in Gold(III) Complexes: Synthesis and Protein Interactions

Inorganics ◽  
2018 ◽  
Vol 7 (1) ◽  
pp. 1 ◽  
Author(s):  
James Beaton ◽  
Nicholas Farrell
Keyword(s):  
Fluorescence Quenching ◽  
Zinc Finger ◽  
Protein Interactions ◽  
Nucleocapsid Protein ◽  
1H Nmr Spectroscopy ◽  
Ionization Mass ◽  
Target Interaction ◽  
Self Association ◽  
A Charge ◽  
First Time

The HIV nucleocapsid protein NCp7 was previously shown to play a number of roles in the viral life cycle and was previously identified as a potential target for small molecule intervention. In this work, the synthesis of the previously unreported complexes [Au(dien)(1MeCyt)]3+, [Au(N-Medien)(1MeCyt)]3+, and [Au(dien)(Cyt)]3+ is detailed, and the interactions of these complexes with the models for NCp7 are described. The affinity for these complexes with the target interaction site, the “essential” tryptophan of the C-terminal zinc finger motif of NCp7, was investigated through the use of a fluorescence quenching assay and by 1H-NMR spectroscopy. The association of [Au(dien)(1MeCyt)]3+ as determined through fluorescence quenching is intermediate between the previously reported DMAP and 9-EtGua analogs, while the associations of [Au(N-Medien)(1MeCyt)]3+ and [Au(dien)(Cyt)]3+ are lower than the previously reported complexes. Additionally, NMR investigation shows that the self-association of relevant compounds is negligible. The specifics of the interaction with the C-terminal zinc finger were investigated by circular dichroism spectroscopy and electrospray-ionization mass spectrometry. The interaction is complete nearly immediately upon mixing, and the formation of AuxFn+ (x = 1, 2, or 4; F = apopeptide) concomitant with the loss of all ligands is observed. Additionally, oxidized dimerized peptide was observed for the first time as a product, indicating a reaction via a charge transfer mechanism.

Assembly states of the nucleosome assembly protein 1 (NAP-1) revealed by sedimentation velocity and non-denaturing MS

2011 ◽  
Vol 436 (1) ◽  
pp. 101-112 ◽  
Author(s):  
Masanori Noda ◽  
Susumu Uchiyama ◽  
Adam R. McKay ◽  
Akihiro Morimoto ◽  
Shigeki Misawa ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Electrostatic Interactions ◽  
Analytical Ultracentrifugation ◽  
Three Dimensional ◽  
Sedimentation Velocity ◽  
Dimensional Structure ◽  
Histone H2a ◽  
Nucleosome Assembly ◽  
Nucleosome Assembly Protein ◽  
Nucleosome Assembly Protein 1

Proteins often exist as ensembles of interconverting states in solution which are often difficult to quantify. In the present manuscript we show that the combination of MS under nondenaturing conditions and AUC-SV (analytical ultracentrifugation sedimentation velocity) unambiguously clarifies a distribution of states and hydrodynamic shapes of assembled oligomers for the NAP-1 (nucleosome assembly protein 1). MS established the number of associated units, which was utilized as input for the numerical analysis of AUC-SV profiles. The AUC-SV analysis revealed that less than 1% of NAP-1 monomer exists at the micromolar concentration range and that the basic assembly unit consists of dimers of yeast or human NAP-1. These dimers interact non-covalently to form even-numbered higher-assembly states, such as tetramers, hexamers, octamers and decamers. MS and AUC-SV consistently showed that the formation of the higher oligomers was suppressed with increasing ionic strength, implicating electrostatic interactions in the formation of higher oligomers. The hydrodynamic shapes of the NAP-1 tetramer estimated from AUC-SV agreed with the previously proposed assembly models built using the known three-dimensional structure of yeast NAP-1. Those of the hexamer and octamer could be represented by new models shown in the present study. Additionally, MS was used to measure the stoichiometry of the interaction between the human NAP-1 dimer and the histone H2A–H2B dimer or H3–H4 tetramer. The present study illustrates a rigorous procedure for the analysis of protein assembly and protein–protein interactions in solution.

Effect of viscosity and dielectric constant variation on fractional fluorescence quenching analysis of coumarin dye in binary solvent mixtures

Luminescence ◽  
2018 ◽  
Vol 33 (5) ◽  
pp. 933-940 ◽  
Author(s):  
P. Bhavya ◽  
Raveendra Melavanki ◽  
Raviraj Kusanur ◽  
Kalpana Sharma ◽  
V. T. Muttannavar ◽  
...  
Keyword(s):  
Dielectric Constant ◽  
Fluorescence Quenching ◽  
Binary Solvent ◽  
Solvent Mixtures ◽  
Binary Solvent Mixtures ◽  
Quenching Analysis ◽  
Coumarin Dye

scholarly journals Humic Acid Promotes the Adsorption of Lead onto PSMPs: Site Energy Distribution Theory and Fluorescence Quenching Analysis

2021 ◽  
Author(s):  
Xiaotian Lu ◽  
Feng Zeng ◽  
Shuyin Wei ◽  
Rui Gao ◽  
Abliz Abdurahman ◽  
...  
Keyword(s):  
Fluorescence Quenching ◽  
Energy Distribution ◽  
Adsorption Capacity ◽  
Distribution Analysis ◽  
Metal Pollutants ◽  
Toxicological Effects ◽  
Adsorption Sites ◽  
Site Energy ◽  
Quenching Analysis ◽  
Heavy Metal Pollutants

Abstract Microplastics (MPs) have a great potential to adsorb heavy metal pollutants such as Pb2+ and the dissolved organic matter(DOM) in the aquatic environment will affect this adsorption behavior. In this study, batch experiments were performed to investigate the adsorption characteristics of Pb2+ onto polystyrene microplastics (PSMPs) in the presence and absence of HA(a kind of representative DOM). The adsorption kinetics of Pb2+ onto PSMPs conformed to the pseudo-second order model, and the adsorption isotherms were well fitted by the Langmuir model. With the increase of HA concentration, the Pb2+ adsorption onto PSMPs increased. Site energy distribution analysis showed that the presence of HA increased the adsorption site energy of PSMPs, thus enhancing the adsorption capacity for Pb2+. The fluorescence quenching analysis of HA further indicated that part of HA were adsorbed onto PSMPs, which increased additional binding sites on the surface of PSMPs. This was attributed to the abundant functional groups that could react with Pb2+ of HA. The pH and ionic strength of solution changed the structure of HA and the adsorption sites of PSMPs, which influenced the adsorption capacity of PSMPs for Pb2+. This study illustrated the effect of HA on the process and mechanism of Pb2+ adsorption onto PSMPs, and provided more information for the evaluation of environmental behavior and toxicological effects of microplastics in aquatic environments.

scholarly journals Author response: Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions

2016 ◽  
Author(s):  
Huaying Zhao ◽  
Yan Fu ◽  
Carla Glasser ◽  
Eric J Andrade Alba ◽  
Mark L Mayer ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Sedimentation Velocity ◽  
Author Response ◽  
High Affinity
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