Application of fluorescence lifetime imaging of enhanced green fluorescent protein to intracellular pH measurements

2008 ◽  
Vol 7 (6) ◽  
pp. 668 ◽  
Author(s):  
Takakazu Nakabayashi ◽  
Hui-Ping Wang ◽  
Masataka Kinjo ◽  
Nobuhiro Ohta
Keyword(s):  
Green Fluorescent Protein ◽  
Intracellular Ph ◽  
Fluorescence Lifetime ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Fluorescence Lifetime Imaging ◽  
Ph Measurements ◽  
Lifetime Imaging ◽  
Green Fluorescent

scholarly journals Intracellular pH measurements made simple by fluorescent protein probes and the phasor approach to fluorescence lifetime imaging

2012 ◽  
Vol 48 (42) ◽  
pp. 5127 ◽  
Author(s):  
Antonella Battisti ◽  
Michelle A. Digman ◽  
Enrico Gratton ◽  
Barbara Storti ◽  
Fabio Beltram ◽  
...  
Keyword(s):  
Intracellular Ph ◽  
Fluorescence Lifetime ◽  
Fluorescent Protein ◽  
Fluorescence Lifetime Imaging ◽  
Ph Measurements ◽  
Lifetime Imaging

Functionalized CdSe/ZnS Quantum Dots for Intracellular pH Measurements by Fluorescence Lifetime Imaging Microscopy

ACS Sensors ◽  
2020 ◽  
Vol 5 (7) ◽  
pp. 2106-2117 ◽  
Author(s):  
Pedro J. Pacheco-Liñán ◽  
Iván Bravo ◽  
María L. Nueda ◽  
José Albaladejo ◽  
Andrés Garzón-Ruiz
Keyword(s):  
Quantum Dots ◽  
Intracellular Ph ◽  
Fluorescence Lifetime ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Ph Measurements ◽  
Lifetime Imaging

scholarly journals Deciphering the Role of Positions 145 and 165 in Fluorescence Lifetime Shortening in the EGFP Variants

Biomolecules ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1547
Author(s):  
Anastasia V. Mamontova ◽  
Aleksander M. Shakhov ◽  
Konstantin A. Lukyanov ◽  
Alexey M. Bogdanov
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescence Lifetime ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Important Determinant ◽  
Physicochemical Characteristics ◽  
High Brightness ◽  
Short Lifetime ◽  
Green Fluorescent

The bright ultimately short lifetime enhanced emitter (BrUSLEE) green fluorescent protein, which differs from the enhanced green fluorescent protein (EGFP) in three mutations, exhibits an extremely short fluorescence lifetime at a relatively high brightness. An important contribution to shortening the BrUSLEE fluorescence lifetime compared to EGFP is provided by the T65G substitution of chromophore-forming residue and the Y145M mutation touching the chromophore environment. Although the influence of the T65G mutation was studied previously, the role of the 145th position in determining the GFPs physicochemical characteristics remains unclear. In this work, we show that the Y145M substitution, both alone and in combination with the F165Y mutation, does not shorten the fluorescence lifetime of EGFP-derived mutants. Thus, the unlocking of Y145M as an important determinant of lifetime tuning is possible only cooperatively with mutations at position 65. We also show here that the introduction of a T65G substitution into EGFP causes complex photobehavior of the respective mutants in the lifetime domain, namely, the appearance of two fluorescent states with different lifetimes, preserved in any combination with the Y145M and F165Y substitutions.

scholarly journals Unravelling the mechanisms controlling heme supply and demand

2021 ◽  
Vol 118 (22) ◽  
pp. e2104008118
Author(s):  
Galvin C.-H. Leung ◽  
Simon S.-P. Fung ◽  
Andrea E. Gallio ◽  
Robert Blore ◽  
Dominic Alibhai ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescence Lifetime ◽  
Fluorescent Protein ◽  
Supply And Demand ◽  
Fluorescence Lifetime Imaging ◽  
Prosthetic Group ◽  
Lifetime Imaging ◽  
Free Heme ◽  
Green Fluorescent ◽  
Cellular Compartments

In addition to heme’s role as the prosthetic group buried inside many different proteins that are ubiquitous in biology, there is new evidence that heme has substantive roles in cellular signaling and regulation. This means that heme must be available in locations distant from its place of synthesis (mitochondria) in response to transient cellular demands. A longstanding question has been to establish the mechanisms that control the supply and demand for cellular heme. By fusing a monomeric heme-binding peroxidase (ascorbate peroxidase, mAPX) to a monomeric form of green-fluorescent protein (mEGFP), we have developed a heme sensor (mAPXmEGFP) that can respond to heme availability. By means of fluorescence lifetime imaging, this heme sensor can be used to quantify heme concentrations; values of the mean fluorescence lifetime (τMean) for mAPX-mEGFP are shown to be responsive to changes in free (unbound) heme concentration in cells. The results demonstrate that concentrations are typically limited to one molecule or less within cellular compartments. These miniscule amounts of free heme are consistent with a system that sequesters the heme and is able to buffer changes in heme availability while retaining the capability to mobilize heme when and where it is needed. We propose that this exchangeable supply of heme can operate using mechanisms for heme transfer that are analogous to classical ligand-exchange mechanisms. This exquisite control, in which heme is made available for transfer one molecule at a time, protects the cell against the toxic effect of excess heme and offers a simple mechanism for heme-dependent regulation in single-molecule steps.

Sign in / Sign up

Export Citation Format

Share Document