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Epidermal growth factor receptor (EGFR) activation by growth factors (GFs) relies on dimerization and allosteric activation of its intrinsic kinase activity, resulting in trans-phosphorylation of tyrosines on its C-terminal tail. Learn more Download Backup and Sync for Mac Download Backup and Sync for Windows. Version 2.0 provides several new features in the user interface together with a real-time correlator for Fluorescence Correlation Spectroscopy (FCS).Access all of your Google Drive content directly from your Mac or PC, without using up disk space. An update of the data acquisition software for the PicoHarp 300 Time-Correlated Single Photon Counting system from PicoQuant is now available.

Conformational movements of these TKD regions will alter the distance and angle between mCitrine and Atto590 resulting in changes in FRET efficiency, which can be quantified by fluorescence lifetime imaging microscopy (FLIM). This hybrid sensor design allows measuring structural changes in different key functional TKD regions relative to the donor. The FFSDP can import both raw data traces and directly exported from To monitor conformational states of the TKD, we engineered multiple FRET-based conformational EGFR sensor variants, in which the donor, monomeric Citrine (mCitrine), was always genetically encoded at the same rigid region of the TKD and genetic code expansion and bioorthogonal labeling chemistry were used to position the acceptor, Atto590, at different, flexible structures of the TKD that change conformation upon activation (Fig. FCS, FCCS, TIR-FCS, PCH, PCMH, FCA, TIFCA and Coincidence analysis are supported. We then show that Y 845 phosphorylation depends on auto-phosphorylation of the C-terminal tail, which creates an autocatalytic loop that amplifies EGFR phosphorylation at low, non-saturating EGF concentrations.FFS Data Processor (FFSDP.exe) free download, latest version 2.3, The FFS Data Processor (FFSDP) software is developed to analyze the data obtained from Fluorescence Fluctuation Spectroscopy measurements. Monitoring conformational transitions together with dimerization and auto-phosphorylation and comparing these readouts to a CONEGI Y845F mutant reveals that an active conformation in monomeric receptors is induced by Y 845 phosphorylation.

B Alignment of active (red PDB: 2J5F) and inactive (cyan PDB: 2GS7) crystal structures of the EGFR TKD. MCitrine is fused to the C-terminal end of the tyrosine kinase domain (TKD) using a coiled-coil linker (dashed line) and Atto590 (red star) is site-specifically attached to the activation loop. A Schematic representation of CONEGI. This project tries to implements MAC (FLASK architecture) on a standard Linux 2.6.x kernel.Design and performance of CONEGI.

Blots were probed with anti-EGFR and anti-Tubulin (left). D Fluorescence images and western blot analysis following SDS-PAGE of HEK293T cell lysates showing Atto590 fluorescence and expression level of EGFR-QG-mCitrine and CONEGIs depending on BCNK. C Relative phosphorylation (PY72/EGFR) of EGFR-mCitrine or EGFR-QG-mCitrine upon EGF stimulation determined by western blot analysis ( nвЂ‰=вЂ‰4 two-way analysis of variance (two-way ANOVA)) (Supplementary Fig.

О¤, fluorescence lifetime of mCitrine. Data points represent individual cells. H Dependency of П„ in CONEGIs and EGFR-QG-mCitrine (QG: nвЂ‰=вЂ‰42 cells 712/713: nвЂ‰=вЂ‰36 713: nвЂ‰=вЂ‰43 730: nвЂ‰=вЂ‰36 737: nвЂ‰=вЂ‰30 843: nвЂ‰=вЂ‰44 851: nвЂ‰=вЂ‰32) on mean Atto590 fluorescence intensity ( F of Atto590) at the PM after 20вЂ‰min labeling. G Mean П„ in CONEGIs and EGFR-QG-mCitrine (QG: nвЂ‰=вЂ‰10 cells 712/713: nвЂ‰=вЂ‰8 713: nвЂ‰=вЂ‰6 730: nвЂ‰=вЂ‰11 737: nвЂ‰=вЂ‰7 843: nвЂ‰=вЂ‰10 851: nвЂ‰=вЂ‰17) at the PM upon tetrazine-Atto590 addition. F Representative fluorescence images of EGFR(BCNK851)-QG-mCitrine upon tetrazine-Atto590 labeling and corresponding П„. E Representative mCitrine and Atto590 fluorescence images of EGFR-QG-mCitrine and CONEGIs in HEK293T cells and corresponding green/magenta overlay.

This site is exposed to the protein surface and not part of the asymmetric as well as the symmetric dimerization interface or regions known to be essential for kinase activity (Supplementary Fig. 1b), we identified a conformationally invariant region at the TKD end, where we inserted mCitrine between amino acids (aa) Q 958 and G 959 (EGFR-QG-mCitrine) 15. 1c: Tukey box plotBy aligning active (red PDB: 2J5F) and inactive (cyan PDB: 2GS7) crystal structures of the EGFR TKD (Fig. Error bars: SEM, except Fig.

We estimated the distances between the QG site that included the maximum linker length and each proposed site of Atto590 attachment. This resulted in the creation of a series of EGFR(TAGXXX)-QG-mCitrine variants, where XXX indicates the amber codon position. We replaced the coding sequences of amino acids K 713 in the kinase loop 2, K 730 and D 737 in the О±C-helix and K 843 and K 851 in the activation loop with an amber codon and also inserted an amber codon between the coding sequence for E 712 and K 713. We then selected three regions (kinase loop 2, О±C-helix, and activation loop) that showed substantial differences between the active and inactive conformation for Atto590 attachment (Fig. 1b, c), which was shown to follow the localization and activity of endogenous EGFR 21, 22. EGFR-QG-mCitrine was fully active and correctly localized as apparent from its similar localization and EGF-induced phosphorylation as compared to C-terminally tagged EGFR (EGFR-mCitrine) (Fig.

This showed that the 851 site yielded the highest (21%) FRET efficiency.

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