生物化学

Fluorescent protein biosensors (FPBs) that turn on—go from dark to bright upon binding their ligands—enable the detection of targets in living cells with high sensitivity and spatial localization. Several approaches exist for creating turn-on FPBs, most notably the method that gave rise to the GCaMP family of genetically encoded calcium indicators. However, it remains challenging to modify these sensors to recognize new ligands. We recently developed adaptable turn-on maturation (ATOM) biosensors, in which target recognition by a small binding domain triggers chromophore maturation in the fluorescent protein to which it is attached. ATOM sensors are advantageous because they are generalizable (by virtue of the monobody and nanobody binding domains) and modular (binding domains and fluorescent proteins of various colors can be mixed and matched for multiplexed imaging), capable of detecting endogenously expressed proteins, and able to function in subcellular compartments including the cytoplasm, nucleus, endoplasmic reticulum, and mitochondria. The protocols herein detail how to design, clone, and screen new ATOM sensors for detecting targets of choice. The starting materials are the genes encoding for a monobody or nanobody and for a cyan, yellow, or red fluorescent protein. We also present general guidelines for creating ATOM sensors using binding domains other than nanobodies and monobodies.

Voltage clamp fluorometry (VCF) is a powerful technique in which the voltage of a cell’s membrane is clamped to control voltage-sensitive membrane proteins while simultaneously measuring fluorescent signals from a protein of interest. By combining fluorescence measurements with electrophysiology, VCF provides real-time measurement of a protein’s motions, which gives insight into its function. This protocol describes the use of VCF to study a membrane protein, the voltage-sensing phosphatase (VSP). VSP is a 3 and 5 phosphatidylinositol phosphate (PIP) phosphatase coupled to a voltage sensing domain (VSD). The VSD of VSP is homologous to the VSD of ion channels, with four transmembrane helices (S1–S4). The S4 contains the gating charge arginine residues that sense the membrane’s electric field. Membrane depolarization moves the S4 into a state that activates the cytosolic phosphatase domain. To monitor the movement of S4, the environmentally sensitive fluorophore tetramethylrhodamine-6-maleimide (TMRM) is attached extracellularly to the S3-S4 loop. Using VCF, the resulting fluorescence signals from the S4 movement measure the kinetics of activation and repolarization, as well as the voltage dependence of the VSD. This protocol details the steps to express VSP in Xenopus laevis oocytes and then acquire and analyze the resulting VCF data. VCF is advantageous as it provides voltage control of VSP in a native membrane while quantitatively assessing the functional properties of the VSD.

Arbuscular mycorrhizal (AM) fungi engage in symbiotic relationships with plants, influencing their phosphate (Pi) uptake pathways, metabolism, and root cell physiology. Despite the significant role of Pi, its distribution and response dynamics in mycorrhizal roots remain largely unexplored. While traditional techniques for Pi measurement have shed some light on this, real-time cellular-level monitoring has been a challenge. With the evolution of quantitative imaging with confocal microscopy, particularly the use of genetically encoded fluorescent sensors, live imaging of intracellular Pi concentrations is now achievable. Among these sensors, fluorescence resonance energy transfer (FRET)-based biosensors stand out for their accuracy. In this study, we employ the Pi-specific biosensor (cpFLIPPi-5.3m) targeted to the cytosol or plastids of Brachypodium distachyon plants, enabling us to monitor intracellular Pi dynamics during AM symbiosis. A complementary control sensor, cpFLIPPi-Null, is introduced to monitor non-Pi-specific changes. Leveraging a semi-automated ImageJ macro for sensitized FRET analysis, this method provides a precise and efficient way to determine relative intracellular Pi levels at the level of individual cells or organelles.

Cytosolic peptide: N-glycanase (PNGase/NGLY1 in mammals), an amidase classified under EC:3.5.1.52, is a highly conserved enzyme across eukaryotes that catalyzes the removal of N-glycans from glycoproteins, converting N-glycosylated asparagine residues into aspartic acid. This enzyme also plays a role in the quality control system for nascent glycoproteins. Despite the development of non-radioisotope-based assay systems such as those using S-alkylated RNase or fluorescent-labeled glycopeptides as substrates, these methods are incompatible with crude enzyme sources, primarily due to the degradation of reaction products by contaminating endogenous proteases. We previously developed an assay system using a 5-carboxyfluorescein-labeled glycosylated cyclo-heptapeptide (5FAM-GCP), a substrate remarkably resistant to endogenous peptidase activity. This system enables the accurate measurement of endogenous NGLY1 activity in various samples, including cell lines, tissues, peripheral blood mononuclear cells, and NGLY1-deficient patient-derived cells, without the interference of proteolytic degradation. We recently advanced this approach by producing a novel fluorescence resonance energy transfer (FRET)-based GCP probe (fGCP) and demonstrated its ability to detect endogenous NGLY1 activity across diverse enzyme sources via fluorescence on multiarray plates. This innovative and straightforward assay now offers reliable disease diagnostics and also allows the measurement of endogenous PNGase/NGLY1 activities across various organisms.

Bioorthogonal chemical reporters are non-native chemical handles introduced into biomolecules of living systems, typically through metabolic or protein engineering. These functionalities can undergo bioorthogonal reactions, such as copper-catalyzed alkyne-azide cycloaddition (CuAAC), with small-molecule probes, enabling the tagging and visualization of biomolecules. This approach has greatly enhanced our understanding of cellular dynamics, enzyme targeting, and protein post-translational modifications. Herein, we report a protocol for preparing protein lysates for click reaction and in-gel fluorescence analysis, exemplified using alk-16, a terminal alkyne-functionalized stearic acid analog, to investigate proteins with fatty acylation. This protocol provides methods for the fluorescent visualization of chemical reporter–labeled proteomes or individual proteins of interest (POIs).

Microscale thermophoresis (MST) is a technique used to measure the strength of molecular interactions. MST is a thermophoretic-based technique that monitors the change in fluorescence associated with the movement of fluorescent-labeled molecules in response to a temperature gradient triggered by an IR LASER. MST has advantages over other approaches for examining molecular interactions, such as isothermal titration calorimetry, nuclear magnetic resonance, biolayer interferometry, and surface plasmon resonance, requiring a small sample size that does not need to be immobilized and a high-sensitivity fluorescence detection. In addition, since the approach involves the loading of samples into capillaries that can be easily sealed, it can be adapted to analyze oxygen-sensitive samples. In this Bio-protocol, we describe the troubleshooting and optimization we have done to enable the use of MST to examine protein–protein interactions, protein–ligand interactions, and protein–nanocrystal interactions. The salient elements in the developed procedures include 1) loading and sealing capabilities in an anaerobic chamber for analysis using a NanoTemper MST located on the benchtop in air, 2) identification of the optimal reducing agents compatible with data acquisition with effective protection against trace oxygen, and 3) the optimization of data acquisition and analysis procedures. The procedures lay the groundwork to define the determinants of molecular interactions in these technically demanding systems.

The assessment of peptide–protein interactions is a pivotal aspect of studying the functionality and mechanisms of various bioactive peptides. In this context, it is essential to employ methods that meet specific criteria, including sensitivity, biocompatibility, versatility, simplicity, and the ability to offer real-time monitoring. In cellular contexts, only a few proteins naturally possess inherent fluorescence, specifically those containing aromatic amino acids, particularly tryptophan. Nonetheless, by covalently attaching fluorescent markers, almost all proteins can be modified for monitoring purposes. Among the early extrinsic fluorescent probes designed for this task, dansyl chloride (DNSC) is a notable option due to its versatile nature and reliable performance. DNSC has been the primary choice as a fluorogenic derivatizing reagent for analyzing amino acids in proteins and peptides for an extended period of time. In our work, we have effectively utilized the distinctive properties of dansylated-calmodulin (D-CaM) for monitoring the interaction dynamics between proteins and peptides, particularly in the context of their association with calmodulin (CaM), a calcium-dependent regulatory protein. This technique not only enables us to scrutinize the affinity of diverse ligands but also sheds light on the intricate role played by calcium in these interactions.

Key features

• Dynamic fluorescence and real-time monitoring: dansyl-modified CaM enables sensitive, real-time fluorescence, providing valuable insights into the dynamics of molecular interactions and ligand binding.

• Selective interaction and stable fluorescent adducts: DNSC selectively interacts with primary amino groups, ensuring specific detection and forming stable fluorescent sulfonamide adducts.

• Versatility in research and ease of identification: D-CaM is a versatile tool in biological research, facilitating identification, precise quantification, and drug assessment for therapeutic development.

• Sensitivity to surrounding alterations: D-CaM exhibits sensitivity to its surroundings, particularly ligand-induced changes, offering subtle insights into molecular interactions and environmental influences.

Graphical overview

Fluorescence emission profiles of dansylated-calmodulin (D-CaM) in different states. Fluorescence emission spectra of D-CaM upon excitation at 320 nm are depicted. Conditions include apo-D-CaM (gray), holo-D-CaM (red), apo-D-CaM bound to peptide (blue), and holo-D-CaM bound to peptide (purple). Corresponding structural representations of D-CaM next to each condition are superimposed on the respective spectra along with the hydrophobicity of the dansyl environment, which increases upon binding of peptide or Ca²⁺ to D-CaM. Upon peptide binding to D-CaM, there is an enhancement in the fluorescent intensity of the spectra; upon Ca²⁺ binding, there is an enhancement of the intensity and a leftward shift of the spectra.

The human pathogenic yeast Candida albicans can attach to epithelial cells or indwelling medical devices to form biofilms. These microbial communities are highly problematic in the clinic as they reduce both sensitivity to antifungal drugs and detection of fungi by the immune system. Amyloid structures are highly organized quaternary structures that play a critical role in biofilm establishment by allowing fungal cells to adhere to each other. Thus, fungal amyloids are exciting targets to develop new antifungal strategies. Thioflavin T is a specific fluorescent dye widely used to study amyloid properties of target proteins in vitro (spectrophotometry) and in vivo (epifluorescence/confocal microscopy). Notably, thioflavin T has been used to demonstrate the ability of Als5, a C. albicans adhesin, to form an amyloid fiber upon adhesion. We have developed a pipeline that allows us to study amyloid properties of target proteins using thioflavin T staining in vitro and in vivo, as well as in intact fungal biofilms. In brief, we used thioflavin T to sequentially stain (i) amyloid peptides, (ii) recombinant proteins, (iii) fungal cells treated or not with amyloid peptides, (iv) fungal amyloids enriched by cell fractionation, and (v) intact biofilms of C. albicans. Contrary to other methods, our pipeline gives a complete picture of the amyloid behavior of target proteins, from in vitro analysis to intact fungal biofilms. Using this pipeline will allow an assessment of the relevance of the in vitro results in cells and the impact of amyloids on the development and/or maintenance of fungal biofilm.

Key features

• Study of amyloid properties of fungal proteins.

• Visualization of the subcellular localization of fungal amyloid material using epifluorescence or confocal microscopy.

• Unraveling of the amyloid properties of target proteins and their physiological meaning for biofilm formation.

• Observation of the presence of amyloid structures with live-cell imaging on intact fungal biofilm using confocal microscopy.

Graphical overview

Biomolecular condensates are membrane-less assemblies of proteins and nucleic acids formed through liquid–liquid phase separation (LLPS). These assemblies are known to temporally and spatially regulate numerous biological activities and cellular processes in plants and animals. In vitro phase separation assay using recombinant proteins represents one of the standard ways to examine the properties of proteins undergoing LLPS. Here, we present a detailed protocol to investigate in vitro LLPS using in vitro expressed and purified recombinant proteins.

Reconstitution of membrane proteins into large unilamellar vesicles is an essential approach for their functional analysis under chemically defined conditions. The orientation of the protein in the liposomal membrane after reconstitution depends on many parameters, and its assessment is important prior to functional measurements. Common approaches for determining the orientation of a membrane-inserted protein are based on limited proteolytic digest, impermeable labeling reagents for specific amino acids, or membrane-impermeable quenchers for fluorescent proteins. Here, we describe a simple site-specific fluorescent assay based on self-labeling enzyme tags to determine the orientation of membrane proteins after reconstitution, exemplified on a reconstituted SNAP-tag plant H⁺-ATPase. This versatile method should benefit the optimization of reconstitution conditions and the analysis of many types of membrane proteins.

Graphical abstract: