We have studied the effect of high hydrostatic pressure and temperature on the steady state fluorescence anisotropy of Green Fluorescent Protein (GFP). We find that the fluorescence anisotropy of GFP at a constant temperature decreases with increasing pressure. At atmospheric pressure, anisotropy decreases with increasing temperature but exhibits a maximum with temperature for pressure larger than 20 MPa. The temperature corresponding to the maximum of anisotropy increases with increasing pressure. By taking into account of the rotational correlation time changes of GFP with the pressure-temperature dependent viscosity of the solvent, we argue that viscosity increase with pressure is not a major contributing factor to the decrease in anisotropy with pressure. The decrease of anisotropy with pressure may result from changes in H-bonding environment around the chromophore.
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http://dx.doi.org/10.1039/d1ra08977c | DOI Listing |
NPJ Biosens
January 2025
Department of Electrical Engineering, University of Victoria, Victoria, BC V8W 3P6 Canada.
The reactivation of heterotrimeric protein phosphatase 2A (PP2A) through small molecule activators is of interest to therapeutic intervention due to its dysregulation, which is linked to chronic conditions. This study focuses on the PP2A scaffold subunit PR65 and a small molecule activator, ATUX-8385, designed to bind directly to this subunit. Using a label-free single-molecule approach with nanoaperture optical tweezers (NOT), we quantify its binding, obtaining a dissociation constant of 13.
View Article and Find Full Text PDFACS Omega
January 2025
Department of Chemistry and Biochemistry, Warren Center for Drug Discovery, The University of Notre Dame, 305 McCourtney Hall, Notre Dame, Indiana 46556, United States.
Selective inhibition of glucose regulated protein 94 (Grp94), the most structurally unique isoform of heat shock protein 90 (Hsp90), has been implicated in the treatment of various disease states, including primary open-angle glaucoma and metastatic cancer. In this study, nine analogues were designed and synthesized by conformationally restricting , a second generation Grp94-selective inhibitor. Conformational constraints were applied to restrict the rotatable bonds and to bias the benzyl moiety into the Grp94 site 1 pocket as well as to reduce the entropic penalty paid upon binding.
View Article and Find Full Text PDFBiomolecular condensates formed via phase separation of proteins and nucleic acids are crucial for the spatiotemporal regulation of a diverse array of essential cellular functions and the maintenance of cellular homeostasis. However, aberrant liquid-to-solid phase transitions of such condensates are associated with several fatal human diseases. Such dynamic membraneless compartments can contain a range of molecular chaperones that can regulate the phase behavior of proteins involved in the formation of these biological condensates.
View Article and Find Full Text PDFRSC Chem Biol
January 2025
Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences Flemingovo n. 2 Prague 6 Czechia
The LutR protein represses the transcription of genes encoding enzymes for the utilization of l-lactate in through binding to a specific DNA region. In this study, we employed oligonucleotide probes modified by viscosity-sensitive tetramethylated thiophene-BODIPY fluorophores to investigate the impact of selected metabolites on the LutR-DNA complex. Our goal was to identify the effector molecule whose binding alters the protein-DNA affinity, thereby enabling gene transcription.
View Article and Find Full Text PDFNan Fang Yi Ke Da Xue Xue Bao
January 2025
Jiangsu Research Institute of Sports Science, Nanjing 210033, China.
Objectives: To investigate the effect of high glucose on macrophage polarization and the role of immune-responsive gene 1 (IRG1) in mediating its effect.
Methods: RAW264.7 cells were transfected with IRG1-overexpressing plasmid or IRG1 siRNA via electroporation and cultured in either normal or high glucose for 72 h to observe the changes in cell viability and morphology using CCK-8 assay and phase contrast microscopy.
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