Combined FCS and PCH Analysis to Quantify Protein Dimerization in Living Cells

Nederveen-Schippers L. M., Pathak P., Keizer-Gunnink I., Westphal A. H., van Haastert P. J. M., Borst J. W., ...More

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol.22, no.14, 2021 (SCI-Expanded) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 22 Issue: 14
  • Publication Date: 2021
  • Doi Number: 10.3390/ijms22147300
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus, Academic Search Premier, CAB Abstracts, EMBASE, Food Science & Technology Abstracts, MEDLINE, Veterinary Science Database, Directory of Open Access Journals
  • Keywords: brightness and diffusion global analysis, Dictyostelium discoideum, dimeric protein, GFP, FK506 binding protein 12, fluorescence correlation spectroscopy, fluorescence fluctuation spectroscopy, photon counting histogram, FLUORESCENCE CORRELATION SPECTROSCOPY, GLOBAL ANALYSIS, RECEPTOR, DISTRIBUTIONS, DIFFUSION, HOMODIMERIZATION, OLIGOMERIZATION, MECHANISM, TRIPLET, NUMBER
  • Süleyman Demirel University Affiliated: No


Protein dimerization plays a crucial role in the regulation of numerous biological processes. However, detecting protein dimers in a cellular environment is still a challenge. Here we present a methodology to measure the extent of dimerization of GFP-tagged proteins in living cells, using a combination of fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis of single-color fluorescence fluctuation data. We named this analysis method brightness and diffusion global analysis (BDGA) and adapted it for biological purposes. Using cell lysates containing different ratios of GFP and tandem-dimer GFP (diGFP), we show that the average brightness per particle is proportional to the fraction of dimer present. We further adapted this methodology for its application in living cells, and we were able to distinguish GFP, diGFP, as well as ligand-induced dimerization of FKBP12 (FK506 binding protein 12)-GFP. While other analysis methods have only sporadically been used to study dimerization in living cells and may be prone to errors, this paper provides a robust approach for the investigation of any cytosolic protein using single-color fluorescence fluctuation spectroscopy.