Abstract
For a complete understanding of biochemical reactions, information on complex stoichiometry is essential. However, measuring stoichiometry is experimentally challenging. Our lab has developed a FRET-based assay to study protein complex stoichiometry in vitro. This assay, also known as Job plot, is set up as a continuous variation of the molar ratio between the two species, kept at constant total concentration. The FRET (Fluorescence Resonance Energy Transfer) between the two fluorescently-labeled proteins is measured and the stoichiometry is inferred from the sample with highest FRET signal. This approach allows us to assess complex stoichiometry in solution.
Keywords: Stoichiometry, FRET, Histones, Fluorescence, Complex formation, Job plot
Background
Each biochemical reaction requires the interaction between two or more cellular components. The stoichiometry of these interactions is an important factor that regulates biochemical reactions in the cell. Experimental determination of complex stoichiometry is therefore critical to fully understand the biochemical and biophysical processes at work within cells. Measuring stoichiometry has been experimentally challenging. For the interaction between large particles that lead to dramatic molecular weight changes, stoichiometry can be inferred by low-resolution structural analysis. These approaches include size-exclusion chromatography, multi-angle light scattering, analytical ultracentrifugation, which are techniques capable of providing accurate molecular weights of the particles. However, these methods require a considerable amount of material and are prone to error when small molecular weight changes are to be observed. We have optimized an assay to measure complex stoichiometry in solution based on FRET (Fluorescence Resonance Energy Transfer). This assay, also known as Job plot (Huang, 1982) can be carried out with considerably less material and it is suitable for studying any complex formation, independent on the size of the two components. In this assay, samples are kept at constant total protein concentration, but with continuous variation of the molar ratio between the two components (Figure 1). The sample with the functional stoichiometry of the complex will display the highest FRET signal. This is a powerful method that enables measurements of complex stoichiometry in solution and among components of any size. Because FRET measurements require both components of the complex to be fluorescently-labeled, a variety of controls are required to exclude potential artifact of the labeling procedure. Ideally single site labeling is advisable in this assay (D’Arcy et al., 2013; Mattiroli et al., 2017), but this is not always possible, as the protein structure may not be known for the components. We suggest performing functional assays with the labeled protein to confirm that the fluorophores do not alter its properties.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Data analysis to calculate the corrected FRET signal is performed as described in Hieb et al. (2012) and Winkler et al. (2012). We attached an example analysis file (Supplemental file 2). In summary:
Notes
Recipes
Acknowledgments
This protocol was adapted from D’Arcy et al., 2013. F.M. is funded by EMBO (ALTF 1267-2013) and the Dutch Cancer Society (KWF 2014-6649). Research in the Luger lab is funded by the Howard Hughes Medical Institute and NIH (GM067777). The authors declare no conflicts of interest or competing interests.
References
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