The photophysical properties of most green fluorescent protein mutants (GFPs) are strongly affected by pH. This effect must be carefully taken into account when using GFPs as fluorescent probes or indicators. Usually, the pH-dependence of GFPs is rationalized on the basis of the ionization equilibrium of the chromophore phenol group. Yet many different mutants show spectral behavior that cannot be explained by ionization of this group alone. In this study, we propose a general model of protonation comprising two ionization sites (2S model). Steady-state optical measurements at different pH and temperature and pH-jump relaxation experiments were combined to highlight the thermodynamic and kinetic properties of paradigmatically different GFP variants. Our experiments support the 2S model. For the case of mutants in which E222 is the second protonation site, thermodynamic coupling between this residue's and the chromophore's ionization reactions was demonstrated. In agreement with the 2S model predictions, X-ray analysis of one of these mutants showed the presence of two chromophore populations at high pH.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1021/bi602646r | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
The Formation and Features of Massive Vacuole Induced by Nutrient Deficiency in Human Embryonic Kidney Cells.
Front Biosci (Landmark Ed)
January 2025
Department of Cardiovascular Medicine, Binzhou Medical University Hospital, 256603 Binzhou, Shandong, China.
Background: Cellular vacuolization is a commonly observed phenomenon under physiological and pathological conditions. However, the mechanisms underlying vacuole formation remain largely unresolved.
Methods: LysoTracker Deep Red probes and Enhanced Green Fluorescent Protein-tagged light chain 3B (LC3B) plasmids were employed to differentiate the types of massive vacuoles.
The Impact of Optical Undersampling on the Ca Signal Resolution in Ca Imaging of Spontaneous Neuronal Activity.
J Integr Neurosci
January 2025
Neuroscience Department, University of Connecticut Health, School of Medicine, Institute for Systems Genomics, Farmington, CT 06030, USA.
Background: In neuroscience, Ca imaging is a prevalent technique used to infer neuronal electrical activity, often relying on optical signals recorded at low sampling rates (3 to 30 Hz) across multiple neurons simultaneously. This study investigated whether increasing the sampling rate preserves critical information that may be missed at slower acquisition speeds.
Methods: Primary neuronal cultures were prepared from the cortex of newborn pups.
Green and Sustainable Spectrofluorometric Determination of Nitrofurantoin in Pure Form and Water Samples Using Functionalized Carbon Quantum Dots.
Luminescence
January 2025
Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt.
The environmental impact of chemicals used in aquaculture, particularly nitrofurantoin, has raised global concern. Nitrofurantoin, a broad-spectrum antimicrobial, is commonly used in aquaculture despite safety risks. Determination of nitrofurantoin in water samples of fish ponds is necessary to ensure the safety and quality of seafood.
View Article and Find Full Text PDFHDAC1 and HDAC2 Are Involved in Influenza A Virus-Induced Nuclear Translocation of Ectopically Expressed STAT3-GFP.
Viruses
December 2024
Department of Microbiology and Immunology, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand.
Influenza A virus (IAV) remains a pandemic threat. Particularly, the evolution and increased interspecies and intercontinental transmission of avian IAV H5N1 subtype highlight the importance of continuously studying the IAV and identifying the determinants of its pathogenesis. Host innate antiviral response is the first line of defense against IAV infection, and the transcription factor, the signal transducer and activator of transcription 3 (STAT3), has emerged as a critical component of this response.
View Article and Find Full Text PDFEstablishment of Recombinant Gene Rabies Virus SRV and Identification of Its Biological Characteristics.
Viruses
December 2024
School of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, China.
Canids act as a crucial intermediary in the transmission of rabies and , serving as co-infection hosts and pathogen carriers for both rabies and hydatid disease (HD) transmitted from animals to humans. Therefore, an effective and efficient bivalent oral vaccine for preventing HD and rabies is urgently required to reduce economic losses in husbandry resulting from rabies and HD. In this study, a full-length plasmid (pcDNA4-NPM+G+EgM123+eGFP+L) carrying the gene and fluorescence reporter genes of eGFP and four auxiliary transfection plasmids of rabies virus SRV (pcDNA4-N, pcDNA4-P, pcDNA4-G, pcDNA-L) were established by reverse genetics approaches and co-transfected to BSR cells by electrotransfection.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!