Draft:Single-molecule pull-down (SiMPull)

Cellular protein interaction approaches are an important part of cellular biology investigations.

Current methods and background on single-molecule analysis

Co-IP methods


 * Co-immunoprecipitation (Co-IP) is a method commonly used to determine interactions between proteins. It involves purifying proteins that are known or suspected to interact, followed by identification of the proteins using techniques such as western blotting or mass spectrometry. Co-IP is considered the gold standard for studying protein interactions, but it can be difficult to accurately determine the number and type of proteins present in a complex. Additionally, the time and multiple steps involved in the process can affect the preservation of in-vivo interactions.

In situ methods


 * In situ imaging methods, including resonance energy transfer, fluorescence correlation spectroscopy, two-hybrid methods, and the bimolecular fluorescence complementation method, are commonly used to study protein interactions. However, these methods cannot be used to study interactions between endogenous proteins and are not able to provide information about the stoichiometry of protein interactions or the presence of heterogeneous interactions.

SiMPull


 * This method is called single-molecule pull-down, or SiMPull, because it involves pulling down physiological macromolecular complexes from cell or tissue extracts directly onto the imaging surface of a single-molecule fluorescence microscope. This method is simple, direct, and sensitive, and allows for the study of cellular protein complexes with single-complex resolution.

General SiMPull application and example

Receptor pull-down


 * The analysis of membrane protein complexes is particularly difficult using conventional methods, which has motivated the development of new approaches. The stoichiometry of these complexes cannot be determined using photobleaching in the cell unless the areal density of the protein complex is low enough for single-molecule detection. SiMPull should be able to detect individual complexes if membrane patches containing one complex can be isolated. As a test, the researchers applied SiMPull to the b2-adrenergic receptor (b2AR), a prototypical G protein-coupled receptor. They transfected HEK293 cells with Flag-tagged YFP-b2AR and solubilized the membrane proteins. They were able to specifically pull down the receptor using antibodies against YFP or Flag. Twenty-nine percent of the traces showed two distinct bleaching steps, indicating a 51% dimer population, assuming 75% of active fluorophores. Less than 3% of the traces showed three or more photobleaching steps. This observation of b2AR homodimerization is consistent with previous studies. To test if b1ARs might form hetero-oligomers with b2ARs, the researchers co-expressed mCherry-tagged b1AR and YFP-tagged b2AR. Using antibodies against mCherry-tagged b1AR, they were able to pull down YFP-tagged b2AR and vice versa. The two fluorophores colocalized with, 42% overlap, consistent with hetero-oligomer formation demonstrating that SiMPull is a powerful tool for receptor pull-down analysis.

Mitochondrial protein pull-down


 * Mitochondrial antiviral signaling (MAVS) is a protein found in the outer membrane of mitochondria that plays a role in the innate immune response. When isolated mitochondrial fractions from cells overexpressing YFP-tagged MAVS were applied to a surface with anti-YFP antibodies, researchers observed bright fluorescent structures. This indicates the presence of several YFP molecules, likely due to the immunoprecipitation of mitochondrial membrane patches or whole mitochondria. When the mitochondrial preparation was pre-solubilized using mild detergent, they observed isolated single YFP spots, 86% of which showed single photobleaching steps, supporting the monomeric state of solubilized MAVS. This suggests that it may be possible to specifically immobilize cellular organelles, or their components using antibodies against suitable marker proteins and perform single-molecule measurements in a physiologically relevant context.

Immunofluorescence detection of single complexes


 * Researchers applied the SiMPull method to the detection of proteins using antibodies, using the mammalian target of rapamycin complex 1 (mTORC1) as a model system. mTORC1 is a signaling complex that regulates cell growth and metabolism in response to nutrient availability. It contains mammalian target of rapamycin (mTOR) and Raptor (RPTOR), which associate with each other at an equimolar ratio. Flag-tagged mTOR and HA-tagged Raptor in HEK293 cells were expressed, pulled down Flag-tagged mTOR using biotinylated Flag antibodies, and detected Raptor using HA antibodies followed by a fluorescently labeled secondary antibody. When both proteins were co-expressed, researchers observed the detection antibody binding as fluorescent spots, while only background levels of fluorescence were detected when only one of the two proteins was expressed. This demonstrates the ability to use antibodies for detection in SiMPull.

Pull-down of endogenous complexes in native tissues


 * Scientists also tested whether SiMPull could be used to detect interactions between endogenous proteins. Endogenous expression of proteins may lead to non-physiological associations between them and detecting interactions between endogenous proteins can be challenging due to their low abundance, high background interactions with other cellular proteins, and the lack of high-affinity antibodies. They used the binding of PKA and AKAP150 as a test case. AKAPs bind to PKA and confine it to specific locations in the cell. They showed that AKAP150 can be co-immunoprecipitated with PKA from mouse brain extract. To make the method more general, a biotin-labeled secondary antibody to immobilize the antibody against the bait protein (PKA) was used and applied mouse brain extract to the surface. When probed for the prey protein (AKAP150) using its primary antibody and a fluorescently labeled secondary antibody, it was observed that ten times more fluorescent spots in the channel with PKA antibody than in the control channel. Impressively, SiMPull required a 20-fold lower sample volume than the corresponding western blot, allowing for the detection of PKA-AKAP binding from mouse heart tissue, which was below the detection limit of the conventional western blot under the same conditions.

SiMPull used for sample preparation

One key advantage of SiMPull is that it allows for the direct observation of protein complexes from fresh cell lysates, bypassing the need for purification procedures. The researchers tested whether SiMPull could be used for functional analysis of pulled-down proteins. They used PcrA, a superfamily 1 helicase, as a test case. PcrA is an ATP-driven motor protein that binds and translocates on single-stranded DNA. The researchers pulled down His6-tagged PcrA from bacterial lysate using anti-His antibodies and added fluorescently labeled DNA molecules to the flow channel. Fluorescent spots due to DNA binding appeared in the flow channel with pulled-down PcrA, while the control channel showed minimal DNA binding. When PcrA binds to a partial duplex DNA with a 59 overhang, it anchors itself to the junction and repetitively reels in the single-stranded DNA. By labeling the DNA with a donor at the tail end and an acceptor at the junction, the researchers were able to observe the reeling-in activity as a gradual increase in fluorescence resonance energy transfer. Once PcrA reaches the end of the single-stranded DNA, it runs off the track and repeats the process, resulting in cyclic increases and decreases in FRET. Eighty-six percent of bound FRET-labeled DNA molecules exhibited repetitive cycling. Increasing the ATP concentration resulted in faster translocation, while in the absence of ATP, DNA remained bound, but no reeling-in activity was observed. The mean translocation time obtained with SiMPull matched well with the data obtained with purified protein. This demonstrates that SiMPull can pull-down functional macromolecules directly from cell extracts for subsequent single-molecule biochemistry in situ.

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