Janghyun Yoo

Real-time single-molecule co-immunoprecipitation analyses reveal cancer-specific Ras signalling dynamics

The conventional co-immunoprecipitation provides static and qualitative information about protein-protein interactions. Lee et al. report real-time imaging of co-immunoprecipitation process with single-molecule resolution, allowing for characterization of the native Ras proteins derived from individual cancers.

Real-time single-molecule coimmunoprecipitation of weak protein-protein interactions

Coimmunoprecipitation (co-IP) analysis is a useful method for studying protein-protein interactions. It currently involves electrophoresis and western blotting, which are not optimized for detecting weak and transient interactions. In this protocol we describe an advanced version of co-IP analysis that uses real-time, single-molecule fluorescence imaging as its detection scheme. Bait proteins are pulled down onto the imaging plane of a total internal reflection (TIR) microscope. With unpurified cells or tissue extracts kept in reaction chambers, we observe single protein-protein interactions between the surface-immobilized bait and the fluorescent protein–labeled prey proteins in real time. Such direct recording provides an improvement of five orders of magnitude in the time resolution of co-IP analysis. With the single-molecule sensitivity and millisecond time resolution, which distinguish our method from other methods for measuring weak protein-protein interactions, it is possible to quantify the interaction kinetics and active fraction of native, unlabeled bait proteins. Real-time single-molecule co-IP analysis, which takes ∼4 h to complete from lysate preparation to kinetic analysis, provides a general avenue for revealing the rich kinetic picture of target protein-protein interactions, and it can be used, for example, to investigate the molecular lesions that drive individual cancers at the level of protein-protein interactions.

PIF1-Interacting Transcription Factors and Their Binding Sequence Elements Determine the in Vivo Targeting Sites of PIF1

In vivo PIF1 targeting to specific promoter sites is determined by PIF1-interacting transcription factors and their binding to G-box coupling sequence elements. The bHLH transcription factor PHYTOCHROME INTERACTING FACTOR1 (PIF1) binds G-box elements in vitro and inhibits light-dependent germination in Arabidopsis thaliana. A previous genome-wide analysis of PIF1 targeting indicated that PIF1 binds 748 sites in imbibed seeds, only 59% of which possess G-box elements. This suggests the G-box is not the sole determinant of PIF1 targeting. The targeting of PIF1 to specific sites could be stabilized by PIF1-interacting transcription factors (PTFs) that bind other nearby sequence elements. Here, we report PIF1 targeting sites are enriched with not only G-boxes but also with other hexameric sequence elements we named G-box coupling elements (GCEs). One of these GCEs possesses an ACGT core and serves as a binding site for group A bZIP transcription factors, including ABSCISIC ACID INSENSITIVE5 (ABI5), which inhibits seed germination in abscisic acid signaling. PIF1 interacts with ABI5 and other group A bZIP transcription factors and together they target a subset of PIF1 binding sites in vivo. In vitro single-molecule fluorescence imaging confirms that ABI5 facilitates PIF1 binding to DNA fragments possessing multiple G-boxes or the GCE alone. Thus, we show in vivo PIF1 targeting to specific binding sites is determined by its interaction with PTFs and their binding to GCEs.

Real-time single-molecule co-immunoprecipitation analyses reveal cancer-specific Ras signaling dynamics

The conventional co-immunoprecipitation provides static and qualitative information about protein-protein interactions. Lee et al. report real-time imaging of co-immunoprecipitation process with single-molecule resolution, allowing for characterization of the native Ras proteins derived from individual cancers.

Correction to “Observing Extremely Weak Protein–Protein Interactions with Conventional Single-Molecule Fluorescence Microscopy”

■ ACKNOWLEDGMENTS This work was supported by the National Creative Research Initiative Program (Center for Single-Molecule Systems Biology to T.-Y.Y., NRF-2011-0018352) and the A3 Foresight Program (to T.-Y.Y., 2013K2A2A6000534, FY2013) through the National Research Foundation of Korea (NRF) funded by the Korean government. This work was also supported by the Institute for Basic Science (IBS; IBS-R026-D1). Addition/Correction