ArticleBiophysical journal2008
Dark states in monomeric red fluorescent proteins studied by fluorescence correlation and single molecule spectroscopy.
Article in Biophysical journal, 2008. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 64 papers.
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64 citing papers in PubMed.
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- Molecular Spies in Action: Genetically Encoded Fluorescent Biosensors Light up Cellular Signals.Chemical reviews · 2024Review
- Diversity of hydrodynamic radii of intrinsically disordered proteins.European biophysics journal : EBJ · 2023Article
- Fluorescent protein lifetimes report densities and phases of nuclear condensates during embryonic stem-cell differentiation.Nature communications · 2023Article
- Quantifying membrane binding and diffusion with fluorescence correlation spectroscopy diffusion laws.Biophysical journal · 2023Article
- Characterization of mApple as a Red Fluorescent Protein for Cryogenic Single-Molecule Imaging with Turn-Off and Turn-On Active Control Mechanisms.The journal of physical chemistry. B · 2023Article
- A comprehensive method to study the DNA's association with lamin and chromatin compaction in intact cell nuclei at super resolution.Nanoscale · 2023Article
- Benchmarking of novel green fluorescent proteins for the quantification of protein oligomerization in living cells.PloS one · 2023Article
- Quantification of Dark Protein Populations in Fluorescent Proteins by Two-Color Coincidence Detection and Nanophotonic Manipulation.The journal of physical chemistry. B · 2022Article
- Networks and Islands of Genome Nano-architecture and Their Potential Relevance for Radiation Biology : (A Hypothesis and Experimental Verification Hints).Results and problems in cell differentiation · 2022Article
- Multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection.eLife · 2021Article
- Elucidation of the Clustered Nano-Architecture of Radiation-Induced DNA Damage Sites and Surrounding Chromatin in Cancer Cells: A Single Molecule Localization Microscopy Approach.International journal of molecular sciences · 2021Article
- Number and Brightness Analysis: Visualization of Protein Oligomeric State in Living Cells.Advances in experimental medicine and biology · 2021Article
- Absolute quantum yield measurements of fluorescent proteins using a plasmonic nanocavity.Communications biology · 2020Article
- Time-correlated single molecule localization microscopy enhances resolution and fidelity.Scientific reports · 2020Article
- Fluorescence Photobleaching as an Intrinsic Tool to Quantify the 3D Expansion Factor of Biological Samples in Expansion Microscopy.ACS omega · 2020Article
- The lipoprotein Pal stabilises the bacterial outer membrane during constriction by a mobilisation-and-capture mechanism.Nature communications · 2020Article
- Quantitative Determination of Dark Chromophore Population Explains the Apparent Low Quantum Yield of Red Fluorescent Proteins.The journal of physical chemistry. B · 2020Article
4 more citing papers are in PubMed but not listed here.
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5 authors.
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Abstract
Monomeric red fluorescent proteins (mRFPs) have become indispensable tools for studying protein dynamics, interactions and functions in the cellular environment. Their emission spectrum can be well separated from other fluorescent proteins, and their monomeric structure preserves the natural function of fusion proteins. However, previous photophysical studies of some RFPs have shown the presence of light-induced dark states that can complicate the interpretation of cellular experiments. In this article, we extend these studies to mRFP1, mCherry, and mStrawberry by means of fluorescence correlation spectroscopy and prove that this light-driven intensity flickering also occurs in these proteins. Furthermore, we show that the flickering in these proteins is pH-dependent. Single molecule spectroscopy revealed reversible transitions from a bright to a dark state in several timescales, even up to seconds. Time-resolved fluorescence spectroscopy showed multiexponential decays, consistent with a "loose" conformation. We offer a structural basis for the fluorescence flickering using known crystal structures and point out that the environment of Glu-215 is critical for the pH dependence of the flickering in RFPs. We apply dual-color fluorescence correlation spectroscopy inside live cells to prove that this flickering can seriously hamper cellular measurements if the timescales of the flickering and diffusion are not well separated.
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