Stoichiometric analysis of protein complexes by cell fusion and single molecule imaging

Abstract

The composition, stoichiometry and interactions of supramolecular protein complexes are a critical determinant of biological function. Several techniques have been developed to study molecular interactions and quantify subunit stoichiometry at the single molecule level. However, these typically require artificially low expression levels or detergent isolation to achieve the low fluorophore concentrations required for single molecule imaging, both of which may bias native subunit interactions. Here we present an alternative approach where protein complexes are assembled at physiological concentrations and subsequently diluted in situ for single-molecule level observations while preserving them in a near-native cellular environment. We show that coupling this dilution strategy with fluorescence correlation spectroscopy permits quantitative assessment of cytoplasmic oligomerization, while stepwise photobleaching and single molecule colocalization may be used to study the subunit stoichiometry of membrane receptors. Single protein recovery after dilution (SPReAD) is a simple and versatile means of extending the concentration range of single molecule measurements into the cellular regime while minimizing potential artifacts and perturbations of protein complex stoichiometry.

Cell-Free Synthesis of a Transmembrane Mechanosensitive Channel Protein into a Hybrid-Supported Lipid Bilayer

“…By assuming a value for the fluorescent fraction (77% of our data), the bleach step histogram can be corrected for "missed" subunits. 40 Our corrected bleach step histogram shows that a number of MscL-GFP protein complexes in the bilayer displayed five discrete bleaching steps (Figure 8). Although we did observe populations of smaller oligomers, with tetramers being the largest fraction, this is typically seen due to bleach steps occurring too close in time to accurately separate and to bleaching that can occur during focusing and locating the field of view.…”

“…To determine the MscL oligomeric state, we used single-molecule bleach step analysis to count the number of subunits of the protein complexes in the field of view for 25% diblock copolymer HSLBs. Subunit counting relies on the detection of the individual bleach steps of fluorescently tagged proteins-of-interest. , For oligomeric protein complexes such as MscL, if all of the fluorescently tagged subunits were fluorescent, the number of photobleaching steps would be equal to the number of subunits per protein complex. However, fluorescent proteins such as GFP can improperly fold and be nonfluorescent, reducing the number of bleach steps observed.…”

“…Although the fraction of misfolded dark proteins can vary with conditions, it is typically ∼20–25% of the total. By assuming a value for the fluorescent fraction (77% of our data), the bleach step histogram can be corrected for “missed” subunits . Our corrected bleach step histogram shows that a number of MscL-GFP protein complexes in the bilayer displayed five discrete bleaching steps (Figure ).…”

“…Photobleaching movies were analyzed by a custom lab software package (ImageC.exe, written in C/C++ under Microsoft Visual Studio). 40 Single-molecule spots were located automatically by successive processing of the summed image stack to locate fluorescent puncta above a threshold based on a userspecified Gaussian fit criterion. For each molecule, a region of interest (ROI) (typically 5 × 5 pixels) centered on the pixel containing the PSF centroid was created and the ROI mean values vs. time (frame) extracted from the stack.…”

“…Photobleaching movies were analyzed by a custom lab software package (ImageC.exe, written in C/C++ under Microsoft Visual Studio) . Single-molecule spots were located automatically by successive processing of the summed image stack to locate fluorescent puncta above a threshold based on a user-specified Gaussian fit criterion.…”