Multiphoton-evoked color change of DsRed as an optical highlighter for cellular and subcellular labeling.

DsRed, a recently cloned red fluorescent protein, has attracted great interest as an expression tracer and fusion partner for multicolor imaging. We report that three-photon excitation (lambda <760 nm) rapidly changes the fluorescence of DsRed from red to green when viewed subsequently by conventional (one-photon) epifluorescence. Mechanistically, three-photon excitation (lambda <760 nm) selectively bleaches the mature, red-emitting form of DsRed, thereby enhancing emission from the immature green form through reduction of fluorescence resonance energy transfer (FRET). The "greening" effect occurs in live mammalian cells at the cellular and subcellular levels, and the resultant color change persists for >30 h without affecting cell viability. This technique allows individual cells, organelles, and fusion proteins to be optically marked and has potential utility for studying cell lineage, organelle dynamics, and protein trafficking, as well as for selective retrieval of cells from a population. We describe optimal parameters to induce the color change of DsRed, and demonstrate applications that show the potential of this optical highlighter.     

Noncytotoxic orange and red/green derivatives of DsRed-Express2 for whole-cell labeling

Background  

Whole-cell labeling is a common application of fluorescent proteins (FPs), but many red and orange FPs exhibit cytotoxicity that limits their use as whole-cell labels. Recently, a tetrameric red FP called DsRed-Express2 was engineered for enhanced solubility and was shown to be noncytotoxic in bacterial and mammalian cells. Our goal was to create derivatives of this protein with different spectral properties.     

A framework for whole-cell mathematical modeling

The default framework for modeling biochemical processes is that of a constant-volume reactor operating under steady-state conditions. This is satisfactory for many applications, but not for modeling growth and division of cells. In this study, a whole-cell modeling framework is developed that assumes expanding volumes and a cell-division cycle. A spherical newborn cell is designed to grow in volume during the growth phase of the cycle. After 80% of the cycle period, the cell begins to divide by constricting about its equator, ultimately affording two spherical cells with total volume equal to twice that of the original. The cell is partitioned into two regions or volumes, namely the cytoplasm (Vcyt) and membrane (Vmem), with molecular components present in each. Both volumes change during the cell cycle; Vcyt changes in response to osmotic pressure changes as nutrients enter the cell from the environment, while Vmem changes in response to this osmotic pressure effect such that membrane thickness remains invariant. The two volumes change at different rates; in most cases, this imposes periodic or oscillatory behavior on all components within the cell. Since the framework itself rather than a particular set of reactions and components is responsible for this behavior, it should be possible to model various biochemical processes within it, affording stable periodic solutions without requiring that the biochemical process itself generates oscillations as an inherent feature. Given that these processes naturally occur in growing and dividing cells, it is reasonable to conclude that the dynamics of component concentrations will be more realistic than when modeled within constant-volume and/or steady-state frameworks. This approach is illustrated using a symbolic whole cell model.     

A rapid whole-cell assay for superoxide dismutase

A procedure is described for the intact-cell assay of superoxide dismutase(s). The technique involves the use of toluene which renders the cells permeable to the necessary components of a photochemical assay for superoxide dismutase. Whole-cell superoxide dismutase activities from a number of procaryotic and eucaryotic microorganisms compare with cell-free activities and with activities reported in the literature. Using this procedure, changing levels of superoxide dismutase are readily monitored under conditions known to modulate superoxide dismutase activity assayed in vitro. In whole cells of Escherichia coli, exogenous methyl viologen causes a marked increase in superoxide dismutase activity, whereas in the cyanobacterium, Microcystis aeruginosa, such treatment leads to a marked, light-dependent loss of whole-cell superoxide dismutase activity.     

Polyubiquitin-regulated DsRed marker for transgenic insects.

Genetic transformation of most insect systems requires dominant-acting markers that do not depend on reverting a mutant phenotype in a host strain, andfor this purpose GFP has proven to be useful in several insect orders. However, detection of multiple transgenes and reporters for gene expression requires the development of new visible markers that can be unambiguously detected when co-expressed with GFP The DsRed fluorescentprotein has spectral characteristics that are most distinct from GFP and GFP variants, and we have explored the use of DsRed as a selectable marker for piggyBac transformation in Drosophila melanogaster and its use as a reporter when co-expressed with GFP. Transformants marked with polyubiquitin-regulated DsRed1 were detected throughout development at a relatively high frequency, and they exhibited brighter fluorescence than transformants marked with EGFP. The use of a Texas Red filter set eliminated detection of EGFP fluorescence and autofluorescence, and DsRed expressedfrom a reporter construct could be unambiguously detected when co-expressed with EGFP DsRed should prove to be a highly efficient marker system for the selection of transformant insects and as a reporter in gene expression studies.     

An enhanced mutant of red fluorescent protein DsRed for double labeling and developmental timer of neural fiber bundle formation.

We developed a new variant of coral-derived red fluorescent protein, DsRed S197Y, which is brighter and essentially free from secondary fluorescence peak. This makes it an ideal reporter for double labeling with green fluorescent protein (GFP). Though purified protein shows only 20% stronger fluorescence emission, culture cells that express DsRed S197Y exhibit a 3-3.5 times higher level of fluorescence than the cells that express wild-type DsRed. The much slower fluorescence maturation of DsRed than that of GFP is a beneficial feature for a fluorescent developmental timer application. When GFP and DsRed S197Y are expressed simultaneously, emissions start at different latency. This provides information about the time after the onset of expression. It reflects the order of cell differentiation if the expression is activated upon differentiation of certain types of cells. We applied this system to the developing brain of Drosophila and visualized, for the first time, the formation order of neural fibers within a large bundle. Our results showed that newly extending fibers of the mushroom body neurons mainly run into the core rather than to the periphery of the existing bundle. DsRed-based timer thus presents an indispensable tool for developmental biology and genetics of model organisms.     

Production and characterization of a noncytotoxic deletion variant of the Aspergillus fumigatus allergen Aspf1 displaying reduced IgE binding

Aspergillus fumigatus is responsible for many allergic respiratory diseases, the most notable of which - due to its severity - is allergic bronchopulmonary aspergillosis. Aspf1 is a major allergen of this fungus: this 149-amino acid protein belongs to the ribotoxin family, whose best characterized member is alpha-sarcin (EC 3.1.27.10). The proteins of this group are cytotoxic ribonucleases that degrade a unique bond in ribosomal RNA impairing protein biosynthesis. Aspf1 and its deletion mutant Aspf1Delta(7-22) have been produced as recombinant proteins; the deleted region corresponds to an exposed beta-hairpin. The conformation of these two proteins has been studied by CD and fluorescence spectroscopy. Their enzymatic activity and cytotoxicity against human rhabdomyosarcoma cells was also measured and their allergenic properties have been studied by using 58 individual sera of patients sensitized to Aspergillus. Aspf1Delta(7-22) lacks cytotoxicity and shows a remarkably reduced IgE reactivity. From these studies it can be concluded that the deleted beta-hairpin is involved in ribosome recognition and is a significant allergenic region.     

A multiscale whole-cell theory for mechanosensitive migration on viscoelastic substrates

Increasing experimental evidence validates that both the elastic stiffness and viscosity of the extracellular matrix regulate mesenchymal cell behavior, such as the rational switch between durotaxis (cell migration to stiffer regions), anti-durotaxis (migration to softer regions), and adurotaxis (stiffness-insensitive migration). To reveal the mechanisms underlying the crossover between these motility regimes, we have developed a multiscale chemomechanical whole-cell theory for mesenchymal migration. Our framework couples the subcellular focal adhesion dynamics at the cell-substrate interface with the cellular cytoskeletal mechanics and the chemical signaling pathways involving Rho GTPase proteins. Upon polarization by the Rho GTPase gradients, our simulated cell migrates by concerted peripheral protrusions and contractions, a hallmark of the mesenchymal mode. The resulting cell dynamics quantitatively reproduces the experimental migration speed as a function of the uniform substrate stiffness and explains the influence of viscosity on the migration efficiency. In the presence of stiffness gradients and absence of chemical polarization, our simulated cell can exhibit durotaxis, anti-durotaxis, and adurotaxis respectively with increasing substrate stiffness or viscosity. The cell moves toward an optimally stiff region from softer regions during durotaxis and from stiffer regions during anti-durotaxis. We show that cell polarization through steep Rho GTPase gradients can reverse the migration direction dictated by the mechanical cues. Overall, our theory demonstrates that opposing durotactic behaviors emerge via the interplay between intracellular signaling and cell-medium mechanical interactions in agreement with experiments, thereby elucidating complex mechanosensing at the single-cell level.     

Activation and growth requirements for cytotoxic and noncytotoxic T lymphocytes

The number and nature of the "signals" required for lymphocyte activation have been so repetitively and academically discussed over the last 15 years that both the readers and the authors appear exhausted by such exercises. Yet, what may be considered to be the essential question, the basis for self-nonself discrimination, remains to be clarified. Since it has been established that clonal expansion and maturation to effector functions are brought about by polyclonally ("immunologically nonspecific") active factors, it is obvious that the crucial "step" in this context is the initial interaction of antigen with specific receptors of immunocompetent lymphocytes. This initial discriminatory event appears to proceed differently on the various cell subsets. We first deal with the mechanism of induction and growth of cytotoxic-T-lymphocyte precursors, and then discuss the inductive requirements leading to proliferation of T helper cells.     

Combination of novel green fluorescent protein mutant TSapphire and DsRed variant mOrange to set up a versatile in planta FRET-FLIM assay

Förster resonance energy transfer (FRET) measurements based on fluorescence lifetime imaging microscopy (FLIM) are increasingly being used to assess molecular conformations and associations in living systems. Reduction in the excited-state lifetime of the donor fluorophore in the presence of an appropriately positioned acceptor is taken as strong evidence of FRET. Traditionally, cyan fluorescent protein has been widely used as a donor fluorophore in FRET experiments. However, given its photolabile nature, low quantum yield, and multiexponential lifetime, cyan fluorescent protein is far from an ideal donor in FRET imaging. Here, we report the application and use of the TSapphire mutant of green fluorescent protein as an efficient donor to mOrange in FLIM-based FRET imaging in intact plant cells. Using time-correlated single photon counting-FLIM, we show that TSapphire expressed in living plant cells decays with lifetime of 2.93 ± 0.09 ns. Chimerically linked TSapphire and mOrange (with 16-amino acid linker in between) exhibit substantial energy transfer based on the reduction in the lifetime of TSapphire in the presence of the acceptor mOrange. Experiments performed with various genetically and/or biochemically known interacting plant proteins demonstrate the versatility of the FRET-FLIM system presented here in different subcellular compartments tested (cytosol, nucleus, and at plasma membrane). The better spectral overlap with red monomers, higher photostability, and monoexponential lifetime of TSapphire makes it an ideal FRET-FLIM donor to study protein-protein interactions in diverse eukaryotic systems overcoming, in particular, many technical challenges encountered (like autofluorescence of cell walls and fluorescence of pigments associated with photosynthetic apparatus) while studying plant protein dynamics and interactions.Single- and dual-color fluorescence imaging with intrinsically fluorescent proteins is increasingly being used to study the expression, targeting, colocalization, turnover, and associations of diverse proteins involved in different plant signal transduction pathways (for review, see Fricker et al., 2006). Concurrent with the use of fluorescence-based cell biology, Förster resonance energy transfer (FRET) has emerged as a convenient tool to study the dynamics of protein associations in vivo. The technique exploits the biophysical phenomenon of nonradiative energy transfer from a donor fluorophore to an appropriately positioned acceptor at a nanometer scale (1–10 nm; Jares-Erijman and Jovin, 2003). In living cells, FRET occurs when two proteins (or different domains within a single protein) fused to suitable donor and acceptor fluorophores physically interact, thus bringing the donor and the acceptor within the favorable proximity for energy transfer (Immink et al., 2002; Bhat et al., 2006). This results in a decrease in the donor''s fluorescence intensity (or quantum yield [QY]) and excited-state lifetime (Gadella et al., 1999). Furthermore, if the acceptor molecule is a fluorophore, then FRET additionally results in an increase in the acceptor''s emission intensity (sensitized emission; Shah et al., 2001; Bhat et al., 2006).However, the exploitation and use of fluorescent marker proteins to study protein trafficking and associations in plants can be problematic because plant cells contain a number of autofluorescent compounds (e.g. lignin, chlorophyll, phenols, etc.) whose emission spectra interfere with that of the most commonly used green or red fluorescent protein fluorophores and/or their spectral variants. For example, lignin fluorescence in roots, vascular tissues, and cell walls of aerial plant parts interferes with imaging at wavelengths between 490 and 620 nm, whereas the chlorophyll autofluorescence in green aerial plant parts is prevalent between 630 and 770 nm (Chapman et al., 2005). Consequently, conventional imaging of GFP and its closest spectral variants (like cyan fluorescent protein [CFP] and yellow fluorescent protein [YFP]) is most likely to be problematic in roots, whereas red-shifted intrinsic fluorescent proteins (including monomeric red fluorescent protein and recently identified spectral variants like mStrawberry and mCherry) may be hard to discriminate in chloroplast-containing aerial tissues (Chapman et al., 2005). The problems get further compounded in FRET assays because the autofluorescence arising from phenols, lignin, and chlorophyll can limit the choice of fluorophores suitable for in planta FRET assays.CFP and YFP have been widely used as a donor-acceptor pair in in planta FRET measurements (Bhat et al., 2006; Dixit et al., 2006). However, in photophysical terms, this pair is less than ideal for FRET imaging. Both have broad excitation and emission spectra with a small Stokes shift (Chapman et al., 2005). Second, QY of CFP (QY = 0.4) is relatively lower than that of YFP (QY = 0.61), and thus a significantly higher (and rather cell damaging) amount of excitation energy is needed to induce FRET (Dixit et al., 2006). Additionally, CFP displays multiexponential lifetimes with a shorter (1.3 ns) and a longer (2.6 ns) component (Becker et al., 2006). Although the deviation from the single-component decay is reasonably small (Tramier et al., 2002; Becker et al., 2006), the shorter CFP lifetime component can erroneously be interpreted as being the result of lifetime reduction due to energy transfer. At the same time, weak or transient protein associations may get masked and thus remain undetected. Whereas the parental wild-type GFP is extremely photostable and shows a monoexponential decay pattern (excited-state lifetime 3.16 ± 0.03 ns; Striker et al., 1999; Volkmer et al., 2000; Shaner et al., 2005), its close spectral overlap with YFP makes it unsuitable as a donor in GFP-YFP FRET experiments. Likewise, wild-type or enhanced GFP (or YFP) as a donor to red-shifted monomers as acceptors is suboptimal because the 488-nm (or 514-nm) laser line commonly used to excite GFP (or YFP) cross excites most of the red monomers (e.g. mOrange, mStrawberry) because of their broad excitation spectra (Zapata-Hommer and Griesbeck, 2003; Shaner et al., 2004).Recently, TSapphire (Q69M/C70P/V163A/S175G; excitation/emission 399/511 nm), a variant of the Sapphire (T203I) mutant of wild-type GFP with improved folding properties and better pH sensitivity, was described (Zapata-Hommer and Griesbeck, 2003). The T203I mutation in TSapphire (and original Sapphire as well) abolishes the 475-nm excitation peak found in the wild-type GFP (Tsien, 1998). TSapphire is efficiently excited below 410 nm, which makes it ideal for studying plant protein dynamics and interactions because, at this wavelength, there is negligible excitation of the autofluorescing chlorophyll pigments. Furthermore, TSapphire also represents a good donor to red monomer acceptors that are negligibly excited at this wavelength (Shaner et al., 2004). Using a purified Zn²⁺ sensor with TSapphire and mOrange as a donor-acceptor pair, Shaner and colleagues demonstrated the ratiometric intramolecular FRET between the two fluorophores in vitro (Shaner et al., 2004). The sensor yielded a 6-fold ratiometric increase (562/514-nm mOrange/TSapphire emission ratio) upon Zn²⁺ binding.However, currently there are no reports demonstrating the application and use of TSapphire and monomeric red-shifted fluorophores as donor-acceptor FRET pairs to probe intermolecular protein-protein interactions in vivo. In this article, we demonstrate in vivo FRET-fluorescence lifetime imaging microscopy (FLIM) between the donor TSapphire and the acceptor mOrange. We show that TSapphire expressed in living plant cells decays with a monoexponential lifetime of 2.93 ± 0.09 ns, which is in agreement with the published lifetime for its parent wild-type GFP (3.2 ns; Striker et al., 1999; Volkmer et al., 2000). Furthermore, we demonstrate intramolecular FRET-FLIM between chimerically linked TSapphire and mOrange (with a 16-amino acid linker in between). When fused to genetically known interacting proteins and expressed in intact living cells, the donor and the acceptor fluorophores show energy transfer in different subcellular compartments indicative of intermolecular protein-protein interactions. These results validate the versatility of the proposed in vivo FRET-FLIM assay based on the donor TSapphire and the acceptor mOrange, which turns out to work with both soluble and membrane proteins.     

A non-toxic fluorogenic dye for mitochondria labeling

Background

Mitochondria, powerhouses of cells, are responsible for many critical cellular functions, such as cell energy metabolism, reactive oxygen species production, and apoptosis regulation. Monitoring mitochondria morphology in live cells temporally and spatially could help with the understanding of the mechanisms of mitochondrial functional regulation and the pathogenesis of mitochondria-related diseases.

Methods

A novel non-cytotoxic fluorogenic compound, AcQCy7, was developed as a mitochondria-specific dye.

Results

AcQCy7 emitted no fluorescent signal outside of cells, but it became fluorescent after intracellular hydrolysis of the acetyl group. The hydrolyzed fluorescent product was well retained in mitochondria, enabling long-lasting fluorescence imaging of mitochondria without cell washing. A 2-day culture study using AcQCy7 showed no sign of cytotoxicity, whereas a commonly used mitochondria-staining probe, Mitochondria Tracker Green, caused significant cell death even at a much lower concentration. Apoptosis-causing mitochondria fission was monitored clearly in real time by AcQCy7.

Conclusions

A simple add-and-read mitochondria specific dye AcQCy7 has been validated in various cell models. Bright mitochondria specific fluorescent signal in treated cells lasted several days without noticeable toxicity.

General Significance

The probe AcQCy7 has been proofed to be a non-toxic agent for long-term mitochondria imaging.     

A new procedure for data reduction in electrophoretic fingerprints of whole-cell proteins

A procedure for the treatment of electrophoretic patterns is presented and discussed using SDS-PAGE patterns of whole-cell proteins of lactic acid bacteria as an example. Complex patterns consisting of two vectors of data [molecular weights (MW), and band intensities] with varying length were transformed in a single vector of fixed length consisting of band intensities accumulated in classes of MW by a logistic weighting function. The procedure performed better than a commercial software (Diversity Database) in clustering the patterns.     

A green fluorescent protein-based whole-cell bioreporter for the detection of phenylacetic acid

Phenylacetic acid (PAA) is produced by many bacteria as an antifungal agent and also appears to be an environmentally toxic chemical. The object of this study was to detect PAA using Pseudomonas putida harboring a reporter plasmid that has a PAA-inducible promoter fused to a green fluorescent protein (GFP) gene. Pseudomonas putida KT2440 was used to construct a green fluorescent protein-based reporter fusion using the paaA promoter region to detect the presence of PAA. The reporter strain exhibited a high level of gfp expression in minimal medium containing PAA; however, the level of GFP expression diminished when glucose was added to the medium, whereas other carbon sources, such as succinate and pyruvate, showed no catabolic repression. Interestingly, overexpression of a paaF gene encoding PAACoA ligase minimized catabolic repression. The reporter strain could also successfully detect PAA produced by other PAA-producing bacteria. This GFP-based bioreporter provides a useful tool for detecting bacteria producing PAA.     

An approach for reducing unwanted oligomerisation of DsRed fusion proteins

Oligomerisation of the red fluorescent protein, DsRed, can interfere with the localisation and function of proteins to which it is fused. We demonstrate an approach that may help to reduce significantly the impact of oligomerisation on the biology of the protein fusion partner. Growth of yeast (Saccharomyces cerevisiae) cells expressing ATP synthase containing subunit gamma-DsRed fusion was compromised relative to control cells. Furthermore, ATP synthase was found to exist as oligomeric structures when isolated under conditions where monomers would normally be present. The compromised growth phenotype was partially reversed and the oligomerisation of the ATP synthase reduced when a non-fluorescent variant of DsRed not fused to another protein was targeted to the mitochondrion in addition to the gamma-DsRed fusion protein. This strategy may also be applicable to the reduction of unwanted interactions between fusion proteins that contain the normally dimeric fluorescent proteins HcRed or Renilla GFP.     

A whole-cell computational model predicts phenotype from genotype

Understanding how complex phenotypes arise from individual molecules and their interactions is a primary challenge in biology that computational approaches are poised to tackle. We report a whole-cell computational model of the life cycle of the human pathogen Mycoplasma genitalium that includes all of its molecular components and their interactions. An integrative approach to modeling that combines diverse mathematics enabled the simultaneous inclusion of fundamentally different cellular processes and experimental measurements. Our whole-cell model accounts for all annotated gene functions and was validated against a broad range of data. The model provides insights into many previously unobserved cellular behaviors, including in vivo rates of protein-DNA association and an inverse relationship between the durations of DNA replication initiation and replication. In addition, experimental analysis directed by model predictions identified previously undetected kinetic parameters and biological functions. We conclude that comprehensive whole-cell models can be used to facilitate biological discovery.     

Novel whole-cell antibiotic biosensors for compound discovery

Cells containing reporters which are specifically induced via selected promoters are used in pharmaceutical drug discovery and in environmental biology. They are used in screening for novel drug candidates and in the detection of bioactive compounds in environmental samples. In this study, we generated and validated a set of five Bacillus subtilis promoters fused to the firefly luciferase reporter gene suitable for cell-based screening, enabling the as yet most-comprehensive high-throughput diagnosis of antibiotic interference in the major biosynthetic pathways of bacteria: the biosynthesis of DNA by the yorB promoter, of RNA by the yvgS promoter, of proteins by the yheI promoter, of the cell wall by the ypuA promoter, and of fatty acids by the fabHB promoter. The reporter cells mainly represent novel antibiotic biosensors compatible with high-throughput screening. We validated the strains by developing screens with a set of 14,000 pure natural products, representing a source of highly diverse chemical entities, many of them with antibiotic activity (6% with anti-Bacillus subtilis activity of 相似文献   

A pretargeted nanoparticle system for tumor cell labeling

Nanoparticle-based cancer diagnostics and therapeutics can be significantly enhanced by selective tissue localization, but the strategy can be complicated by the requirement of a targeting ligand conjugated on nanoparticles, that is specific to only one or a limited few types of neoplastic cells, necessitating the development of multiple nanoparticle systems for different diseases. Here, we present a new nanoparticle system that capitalizes on a targeting pretreatment strategy, where a circulating fusion protein (FP) selectively prelabels the targeted cellular epitope, and a biotinylated iron oxide nanoparticle serves as a secondary label that binds to the FP on the target cell. This approach enables a single nanoparticle formulation to be used with any one of existing fusion proteins to bind a variety of target cells. We demonstrated this approach with two fusion proteins against two model cancer cell lines: lymphoma (Ramos) and leukemia (Jurkat), which showed 72.2% and 91.1% positive labeling, respectively. Notably, TEM analysis showed that a large nanoparticle population was endocytosed via attachment to the non-internalizing CD20 epitope.     

Optimization and 13CH3 methionine labeling of a signaling competent neurotensin receptor 1 variant for NMR studies

Neurotensin is a 13-residue peptide that acts as a neuromodulator of classical neurotransmitters such as dopamine and glutamate in the mammalian central nervous system, mainly by activating the G protein-coupled receptor (GPCR), neurotensin receptor 1 (NTS₁). Agonist binding to GPCRs shifts the conformational equilibrium of the transmembrane helices towards distinct, thermodynamically favorable conformations that favor effector protein interactions and promotes cell signaling. The introduction of site specific labels for NMR spectroscopy has proven useful for investigating this dynamic process, but the low expression levels and poor stability of GPCRs is a hindrance to solution NMR experiments. Several thermostabilized mutants of NTS₁ have been engineered to circumvent this, with the crystal structures of four of these published. The conformational dynamics of NTS₁ however, has not been thoroughly investigated with NMR. It is generally accepted that stabilized GPCRs exhibit attenuated signaling, thus we thoroughly characterized the signaling characteristics of several thermostabilized NTS₁ variants to identify an optimal variant for protein NMR studies. A variant termed enNTS₁ exhibited the best combination of signaling capability and stability upon solubilization with detergents. enNTS₁ was subsequently labeled with ¹³CH₃-methionine in E. coli and purified to homogeneity in the absence of bound ligands. Using solution NMR spectroscopy we observed several well dispersed ¹³CH₃-methionine resonances, many of which exhibited chemical shift changes upon the addition of the high affinity agonist peptide, NT8-13. Thus, enNTS₁ represents a novel tool for investigating ligand induced conformational changes in NTS₁ to gain insights into the molecular mechanisms underlying neurotensin signaling.