Influence of FRET and fluorescent protein maturation on the quantification of binding affinity with dual-channel fluorescence cross-correlation spectroscopy
| Autor: | Jesse Goyette, Rashmi R. Pillai, Varun K. A. Sreenivasan, Matthew S. Graus, Zhengmin Yang, Katharina Gaus |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
0303 health sciences
Fluorescence-lifetime imaging microscopy Chemistry 01 natural sciences Fluorescence Atomic and Molecular Physics and Optics Fluorescence spectroscopy Article 010309 optics 03 medical and health sciences Crosstalk (biology) Membrane Förster resonance energy transfer 0103 physical sciences Biophysics Fluorescence cross-correlation spectroscopy Spectroscopy 030304 developmental biology Biotechnology |
| Zdroj: | Biomed Opt Express |
| ISSN: | 2156-7085 |
| Popis: | Protein-protein interactions at the plasma membrane mediate transmembrane signaling. Dual-channel fluorescence cross-correlation spectroscopy (dc-FCCS) is a method with which these interactions can be quantified in a cellular context. However, factors such as incomplete maturation of fluorescent proteins, spectral crosstalk, and fluorescence resonance energy transfer (FRET) affect quantification. Some of these can be corrected or accounted for during data analysis and/or interpretation. Here, we experimentally and analytically demonstrate that it is difficult to correct the error caused due to FRET when applying dc-FCCS to measure binding affinity or bound molecular concentrations. Additionally, the presence of dark fluorescent proteins due to incomplete maturation introduces further errors, which too cannot be corrected in the presence of FRET. Based on simulations, we find that modalities such as pulse-interleaved excitation FCCS do not eliminate FRET-induced errors. Finally, we demonstrate that the detrimental effect of FRET can be eliminated with careful experimental design when applying dc-FCCS to quantify protein-protein interactions at the plasma membrane of living cells. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|