Biochemistry

Regulated IRE1-dependent decay (RIDD) is a critical cellular mechanism mediated by the endoplasmic reticulum (ER) stress sensor IRE1α, which cleaves a variety of RNA targets to regulate ER homeostasis. Current in vitro assays to study IRE1α activity largely rely on synthetic or in vitro transcribed RNA substrates, which may not fully replicate the physiological complexities of native RNA molecules. Here, we present a comprehensive protocol to assess IRE1α-dependent RNA cleavage activity using total RNA isolated directly from mouse tissues. This protocol provides a step-by-step guide for tissue collection, RNA isolation, an ex vivo RIDD assay, cDNA synthesis, and subsequent RT-PCR analysis of target mRNA cleavage products. Key reagents include active IRE1α protein, the RIDD-specific inhibitor 4μ8C, and target-specific primers for RIDD-regulated genes such asBloc1s1 and Col6a1. Quantitative assessment is achieved using agarose gel electrophoresis and imaging software. This methodology enables the study of IRE1α's RNA cleavage activity under conditions that closely mimic in vivo environments, providing a more physiologically relevant approach to understanding the role of RIDD in cellular and tissue-specific contexts.

Studying G protein-coupled receptor (GPCR) activation of heterotrimeric G proteins is crucial for understanding diverse physiological processes and developing novel therapeutics. Traditional methods to assay GPCR activation of G proteins, including assays of second messengers and biosensors, involve complex or indirect procedures. However, second messengers like cAMP and calcium are not direct readouts of GPCR activity due to signaling crosstalk, while biosensors can have undesired consequences due to structural alteration caused by fluorescent protein insertion. Here, we present a streamlined protocol employing GST-tagged bait proteins and epitope-embedded Gα subunits to achieve direct monitoring of Gα activity within cells. This method involves purification of GST-tagged bait constructs from bacteria and subsequent direct interaction studies with GluGlu-tagged Gα proteins expressed in any human cells of interest by including GST-tagged bait proteins in the cell lysis buffer. The approach enables sensitive detection of activated Gα within cells following extracellular stimulation. Advantages of this protocol include high sensitivity, enhanced monitoring of GPCR signaling dynamics under physiologically relevant conditions with minimum alteration in Gα, and the ability to distinguish between highly homologous isoforms within the same Gα family.

Plants rely on metabolite regulation of proteins to control their metabolism and adapt to environmental changes, but studying these complex interaction networks remains challenging. The proteome integral solubility alteration (PISA) assay, a high-throughput chemoproteomic technique, was originally developed for mammalian systems to investigate drug targets. PISA detects changes in protein stability upon interaction with small molecules, quantified through LC–MS. Here, we present an adapted PISA protocol for Arabidopsis thaliana chloroplasts to identify potential protein interactions with ascorbate. Chloroplasts are extracted using a linear Percoll gradient, treated with multiple ascorbate concentrations, and subjected to heat-induced protein denaturation. Soluble proteins are extracted via ultracentrifugation, and proteome-wide stability changes are quantified using multiplexed LC–MS. We provide instructions for deconvolution of LC–MS spectra and statistical analysis using freely available software. This protocol enables unbiased screening of protein regulation by small molecules in plants without requiring prior knowledge of interaction partners, chemical probe design, or genetic modifications.

Antibody purification is a fundamental technology for therapeutic and diagnostic applications. While traditional methods like ammonium sulfate precipitation and polyethylene glycol precipitation are cost-effective, they often result in low purity and require multiple purification steps. Protein A–based affinity chromatography, the gold standard for antibody purification, provides high specificity but can be further improved to increase loading capacity and reduce costs. In this protocol, we introduce a novel approach for purifying high-quality, high-purity antibodies from complex samples using SpyFixer/Z domain–modified resin. This method utilizes Spy chemistry for site-specific immobilization of the Z domain of Protein A, significantly enhancing antibody loading capacity up to 200 mg/mL resin and ensuring stable, durable immobilization. Using this protocol, we achieved >90% purity of human immunoglobulin G (hIgG) from diverse sources, including E. coli cell lysates, human serum, and industrial fermentation broth. The SpyFixer/Z domain–modified resin protocol is simple, cost-effective, and offers a robust, scalable solution for efficient antibody purification in bioengineering applications. This immobilization scheme based on Spy chemistry can also be extended to other immunoglobulin-binding proteins, such as Protein G and Protein L, to develop high-efficiency affinity resins.

Enteroviruses are abundant pathogens of humans and animals. Their replication is strictly dependent on the conserved, viral AAA+ ATPase 2C. 2C is an oligomerizing, peripheral membrane protein, and its low solubility as recombinant protein has hampered functional studies of the full-length, recombinant protein bound to a membrane. Here, we describe a modification of the classical, ultracentrifugation-based liposome flotation assay optimized to study the interaction of recombinant 2C with membranes and the functions of membrane-bound, full-length recombinant 2C. The assay takes advantage of the high solubility of recombinant 2C while fused to a maltose-binding protein. Removing this solubility-enhancing tag by specific protease cleavage in the presence of liposomes allows 2C to associate with membranes prior to aggregating. Fluorophore labeling of protein and liposomes allows rapid and precise quantitation of 2C’s association with membranes. This assay is adaptable to any peripheral membrane protein that can be fluorophore-labeled and expressed as a solubility-enhancing fusion protein.

Myosin-5a (Myo5a) is an actin-dependent molecular motor that recognizes a diverse range of cargo proteins through its tail domain, playing a crucial role in the transport and localization of various organelles within the cell. We have identified a new interaction between Myo5a and its cargo protein melanophilin (Mlph), i.e., the interaction between the middle tail domain of Myo5a (Myo5a-MTD) and the actin-binding domain of Mlph (Mlph-ABD), by GST pulldown assay. We then intend to obtain the dissociation constant between Myo5a-MTD and Mlph-ABD using isothermal titration calorimetry (ITC) or microscale thermophoresis (MST), both of which are two commonly used methods for determining quantitative data on protein interactions. The advantages of MST over ITC include less protein usage, shorter operation time, and higher sensitivity. In this protocol, we present a method for using MST to determine the dissociation constants of Myo5a-MTD and Mlph-ABD, which were purified through overexpression in bacteria using affinity chromatography. The dissociation constant values obtained directly reflect the binding strength between these two proteins and provide a foundation for the isolation and purification of the complex in the future.

Different research methods aim to clarify the intracellular trafficking of target proteins or unknown pathways. Currently, existing methods are mostly complex and expensive, requiring expert knowledge. Detailed microscopy for protein co-localization detection or omic technologies, which provide holistic network data, are elaborate, mostly complex, and expensive to apply. Our protocol illustrates a method to track a target protein by detecting expression changes of user-selected marker proteins that directly or indirectly interact with the target. Modulation of protein expression indicates interactions between the target and marker protein. Even without co-localization analysis, the results of the protein expression change are the first insights into the target's fate. Moreover, the use of the cell-sonar is straightforward and affordable, and the results are rapidly available. Furthermore, this method could also be used to determine if and how pathways are affected by compounds added to the cells. In conclusion, our method is adaptable to a wide range of proteins, easy to apply, inexpensive, and expandable with substances that affect proteins.

Nicotinic acetylcholine receptors (nAChRs) are a family of ligand-gated ion channels expressed in nervous and non-nervous system tissue important for memory, movement, and sensory processes. The pharmacological targeting of nAChRs, using small molecules or peptides, is a promising approach for the development of compounds for the treatment of various human diseases including inflammatory and neurogenerative disorders such as Alzheimer’s disease. Using the Aplysia californica acetylcholine binding protein (Ac-AChBP) as an established structural surrogate for human homopentameric α7 nAChRs, we describe an innovative protein painting mass spectrometry (MS) method that can be used to identify interaction sites for various ligands at the extracellular nAChR site. We describe how the use of small molecule dyes can be optimized to uncover contact sites for ligand–protein interactions based on MS detection. Protein painting MS has been recently shown to be an effective tool for the identification of residues within Ac-AChBP involved in the binding of know ligands such as α-bungarotoxin. This strategy can be used with computational structural modeling to identify binding regions involved in drug targeting at the nAChR.

Surface Plasmon Resonance (SPR) is a label-free optical technique to assess protein–protein interaction kinetics and affinities in a real-time setting. Traditionally, Biacore SPR employs a continuous film of gold to detect any change in the angle of re-emitted light when the refractive index of a ligand conjugated to the flat gold surface is altered by its interaction with a local analyte. In contrast, the Nicoya Lifesciences’ OpenSPR technology uses gold nanoparticles to detect small changes in the absorbance peak wavelength of a conjugated ligand after its engagement by an analyte. Specifically, when broadband white light is shone onto the gold nanoparticles, it produces a strong resonance absorbance peak corresponding to the refractive index of a ligand conjugated to the surface of gold nanoparticles. Upon its interaction with an analyte, however, the absorbance wavelength peak of the conjugated ligand will be changed and timely recorded as sensorgrams of dynamic ligand–analyte interactions. Thus, the improvement in the detection method (from traditional detection of changes in the angle of re-emitted light to the contemporary detection of changes in the wavelength of the absorbance peak) features OpenSPR as a cost-effective and user-friendly technique for in-depth characterization of protein–protein interactions. Here, we describe the detailed method that we used to characterize procathepsin L (pCTS-L) interactions with two putative pattern recognition receptors (TLR4 and RAGE) using the 1st generation of Nicoya Lifesciences’ OpenSPR instrument with a 1-channel detection.

Key features

• Nicoya OpenSPR is a benchtop small-size equipment that provides in-depth label-free binding kinetics and affinity measurement for protein–protein interactions in real-time fashion.

• This technology is relatively intuitive and user-friendly for scientists at any skill level.

• OpenSPR sensors employ nanotechnology to reduce the cost of manufacturing complex optical hardware and Sensor Chips, and similarly reduce the consumption of precious analyte samples.

• The manufacturer provides online training for OpenSPR (Catalog: TRAIN-REMOTE) and TraceDrawer (Catalog: TRAIN-TD) to customer scientists.

Integral membrane proteins are an important class of cellular proteins. These take part in key cellular processes such as signaling transducing receptors to transporters, many operating within the plasma membrane. More than half of the FDA-approved protein-targeting drugs operate via interaction with proteins that contain at least one membrane-spanning region, yet the characterization and study of their native interactions with therapeutic agents remains a significant challenge. This challenge is due in part to such proteins often being present in small quantities within a cell. Effective solubilization of membrane proteins is also problematic, with the detergents typically employed in solubilizing membranes leading to a loss of functional activity and key interacting partners. In recent years, alternative methods to extract membrane proteins within their native lipid environment have been investigated, with the aim of producing functional nanodiscs, maintaining protein–protein and protein–lipid interactions. A promising approach involves extracting membrane proteins in the form of styrene maleic acid lipid particles (SMALPs) that allow the retention of their native conformation. This extraction method offers many advantages for further protein analysis and allows the study of the protein interactions with other molecules, such as drugs. Here, we describe a protocol for efficient SMALP extraction of functionally active membrane protein complexes within nanodiscs. We showcase the method on the isolation of a low copy number plasma membrane receptor complex, the nicotinic acetylcholine receptor (nAChR), from adult Drosophila melanogaster heads. We demonstrate that these nanodiscs can be used to study native receptor–ligand interactions. This protocol can be applied across many biological scenarios to extract the native conformations of low copy number integral membrane proteins.