The Binding of Fluorophores to Proteins Depends on the Cellular Environment

Kim, Yun Kyung; Lee, Jung Hoon; Bi, Xinyan; Ha, Hyung‐Ho; Ng, Sum Huan; Ahn, Young‐Hoon; Lee, Jae‐Jung; Wagner, Bridget K.; Clemons, Paul A.; Chang, Young‐Tae

doi:10.1002/ange.201007626

Cited by 11 publications

(7 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The modification, however, may change the biological activities of the compounds and the in vitro conditions for binding may bring a false target which is different from the actual in vivo target (7). In contrast, the intrinsic fluorescence property of our DOFL compounds makes it possible to track the targets without any modification from the stained living cells through all processes for target identification once they bind strongly to the target molecules.…”

mentioning

confidence: 99%

Neural stem cell specific fluorescent chemical probe binding to FABP7

Yun

Leong

Zhai

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Fluorescent small molecules have become indispensable tools for biomedical research along with the rapidly developing optical imaging technology. We report here a neural stem cell specific boron-dipyrromethane (BODIPY) derivative compound of designation red 3 (CDr3), developed through a high throughput/content screening of in-house generated diversity oriented fluorescence library in stem cells at different developmental stages. This novel compound specifically detects living neural stem cells of both human and mouse origin. Furthermore, we identified its binding target by proteomic analysis as fatty acid binding protein 7 (FABP7), also known as brain lipid binding protein) which is highly expressed in neural stem cells and localized in the cytoplasm. CDr3 will be a valuable chemical tool in the study and applications of neural stem cells.

show abstract

mentioning

confidence: 99%

Neural stem cell specific fluorescent chemical probe binding to FABP7

Yun

Leong

Zhai

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…However, the specific interactions between bioactive small molecules and target proteins in cell lysates can differ from those in live cells as a result of the extremely high concentration of proteins inside live cells, as well as the nonspecific attractive and repulsive interactions that occur in live cells with many macromolecules, including proteins, nucleotides, lipids, and metabolites 9. Chang and co‐workers reported the environment‐dependent covalent labeling of an organic fluorophore to target proteins, but they did not focus on the functional modulation of target proteins 10. Yao and co‐workers successfully demonstrated the in vitro and in cell target identification of known bioactive small molecules 11.…”

mentioning

confidence: 99%

Discovery and Target Identification of an Antiproliferative Agent in Live Cells Using Fluorescence Difference in Two‐Dimensional Gel Electrophoresis

Park

2012

Angewandte Chemie

View full text Add to dashboard Cite

Ziel gefunden: Durch Fluoreszenzdifferenz in der 2D‐Gelelektrophorese (FITGE) können Wechselwirkungen zwischen Proteinen und Wirkstoffmolekülen in intakter zellulärer Umgebung beobachtet werden. Wo konventionelle Methoden keinen Erfolg hatten, gelang mit FITGE die Identifizierung des Zielproteins einer antiproliferativen Verbindung in lebenden Zellen durch Differenzierung zwischen spezifischer und unspezifischer Bindung von Photoaffinitätssonden.

show abstract

“…Based on the charged nature of the dye, it most likely accumulates at the matrix side of the inner membrane, or within the inner membrane, bound to its targets, including ALDH2, one of known mitochondrial target of MTY [18]. In this respect, the binding of MTY might also depend on the environment provided by the mitochondria (as pH, density and ionic strength) and may vary according to the cell types and their metabolic status [23]. Thus, caution should be taken before extrapolating the measured temperature of 50 • C to the whole mitochondrial network and to the several intra mitochondrial compartments.…”

Section: Discussionmentioning

confidence: 99%

“…Accordingly, MTY accumulates in the mitochondria attracted by the negative mitochondrial membrane potential (MMP). However, after its initial membrane potential-dependent uptake, MTY binds to its targets within the mitochondria, including the mitochondrial ALDH2 [18,23] resulting in retention of the dye in the mitochondria and consequently making it insensitive to mitochondrial potential variations. Thus the fluorescent signal of MTY is unaffected by the confounding effects of fluorescence quenching and is therefore solely influenced by changes in the surrounding temperature.…”

Section: Fully Energized Mitochondria In Hela and Hek Cells Increase mentioning

confidence: 99%

Pitfalls in Monitoring Mitochondrial Temperature Using Charged Thermosensitive Fluorophores

et al. 2020

View full text Add to dashboard Cite

Mitochondria are the source of internal heat which influences all cellular processes. Hence, monitoring mitochondrial temperature provides a unique insight into cell physiology. Using a thermosensitive fluorescent probe MitoThermo Yellow (MTY), we have shown recently that mitochondria within human cells are maintained at close to 50 °C when active, increasing their temperature locally by about 10 °C. Initially reported in the HEK293 cell line, we confirmed this finding in the HeLa cell line. Delving deeper, using MTY and MTX (MitoThermo X), a modified version of MTY, we unraveled some caveats related to the nature of these charged fluorophores. While enabling the assessment of mitochondrial temperature in HEK and HeLa cell lines, the reactivity of MTY to membrane potential variations in human primary skin fibroblasts precluded local temperature monitoring in these cells. Chemical modification of MTY into MTX did not result in a temperature probe unresponsive to membrane potential variations that could be universally used in any cell type to determine mitochondrial temperature. Thus, the cell-type dependence of MTY in measuring mitochondrial temperature, which is likely due to the variable binding of this dye to specific internal mitochondrial components, should imply cautiousness while using these nanothermometers for mitochondrial temperature analysis.

show abstract

The Binding of Fluorophores to Proteins Depends on the Cellular Environment

Abstract: Erleuchtet! Um intrazelluläre Binder für myotubenspezifische Sonden zu untersuchen, wurde das thiolreaktive Derivat CDy2 synthetisiert. In lebenden Zellen reichert sich dieses Derivat selektiv in Mitochondrien an und markiert seine Bindungspartner kovalent.

Cited by 11 publications

References 24 publications

Neural stem cell specific fluorescent chemical probe binding to FABP7

Neural stem cell specific fluorescent chemical probe binding to FABP7

Discovery and Target Identification of an Antiproliferative Agent in Live Cells Using Fluorescence Difference in Two‐Dimensional Gel Electrophoresis

Pitfalls in Monitoring Mitochondrial Temperature Using Charged Thermosensitive Fluorophores

Contact Info

Product

Resources

About