Journal Article

Probing cellular protein complexes using single-molecule pull-down

Ha, Taekjip, Arauz, Edwin, Ragunathan, Kaushik, Xiang, Yang K., Liu, Ruijie, Ramani, Biswarathan, Park, Jeehae, Jain, Ankur, Ishitsuka, Yuji, Chen, Jie

2011

Overview

Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here we describe a single-molecule pull-down (SiMPull) assay that combines the principles of a conventional pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signalling proteins found in the cytosol, membrane and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled-down proteins are functional and are used, without further processing, for single-molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analysing protein assemblies in biological pathways. Protein complexes at single-molecule resolution Analysis of protein interactions is crucial for understanding cellular function and regulation. Here, Taekjip Ha and colleagues develop a novel method for elucidating the identity and stoichiometry of protein complexes from cells and tissues at single-molecule resolution. The method, called single molecule pull-down or SiMPull, can discriminate between multiple association states of a protein, and simultaneously allows determination of complex stoichiometry through photobleaching step analysis. The potential of the assay is demonstrated in a variety of contexts, including endogenous proteins from tissue extracts, organelles and membrane proteins.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 631/1647 / 631/45/612 / 631/57/2265 / Animal tissues / Bacterial Proteins - analysis / Bacterial Proteins - genetics / Bacterial Proteins - metabolism / Biological and medical sciences / Cell Extracts - chemistry / Color / Cyclic AMP-Dependent Protein Kinases - analysis / Cyclic AMP-Dependent Protein Kinases - genetics / Cyclic AMP-Dependent Protein Kinases - metabolism / Diverse techniques / DNA Helicases - analysis / DNA Helicases - metabolism / Fluorescence microscopy / Fluorescence Resonance Energy Transfer / Fluorescent Antibody Technique / Fundamental and applied biological sciences. Psychology / HEK293 Cells / Humanities and Social Sciences / Humans / Immunoprecipitation - methods / Kinases / Labeling / Luminescent Proteins - analysis / Luminescent Proteins - genetics / Luminescent Proteins - metabolism / Mass spectrometry / Methods / Microscopy, Fluorescence / Mitochondrial Proteins - analysis / Mitochondrial Proteins - metabolism / Molecular and cellular biology / multidisciplinary / Multiprotein Complexes - analysis / Multiprotein Complexes - chemistry / Multiprotein Complexes - isolation & purification / Multiprotein Complexes - metabolism / Photobleaching / Protein Binding / Protein Interaction Mapping - methods / Proteins / Receptors, Adrenergic, beta - analysis / Receptors, Adrenergic, beta - metabolism / Science / Science (multidisciplinary) / Tissue Extracts - chemistry / Tissue Extracts - metabolism