Extrinsic Fluorescent Probe Research Articles

Biophysical Studies of Amyloid-Binding Fluorophores to Tau AD Core Fibrils Formed without Cofactors.

Tau is an intrinsically disordered protein involved in several neurodegenerative diseases where a common hallmark is the appearance of tau aggregates in the brain. One common approach to elucidate the mechanisms behind the aggregation of tau has been to recapitulate in vitro the self-assembly process in a fast and reproducible manner. While the seeding of tau aggregation is prompted by negatively charged cofactors, the obtained fibrils are morphologically distinct from those found in vivo. The Tau AD core fragment (TADC, tau 306-378) has emerged as a new model and potential solution for the cofactor-free in vitro aggregation of tau. Here, we use TADC to further study this process combining multiple amyloid-detecting fluorophores and fibril bioimaging. We confirmed by transmission electron microscopy that this fragment forms fibrils after quiescent incubation at 37 °C. We then employed a panel of eight amyloid-binding fluorophores to query the formed species by acquiring their emission spectra. The results obtained showed that nearly all dyes detect TADC self-assembled species. However, the successful monitoring of TADC aggregation kinetics was limited to three fluorophores (X-34, Bis-ANS, and pFTAA) which yielded sigmoidal curves but different aggregation half-times, hinting to different species being detected. Altogether, this study highlights the potential of using multiple extrinsic fluorescent probes, alone or in combination, as tools to further clarify mechanisms behind the aggregation of amyloidogenic proteins.

Inhibition kinetics and mechanism of genistein against α‐glucosidase

AbstractSeveral studies have documented the effective inhibition of genistein, a component in soybeans against α‐glucosidase. However, the details of the inhibition mechanism and kinetics remain unfulfilled. Here, the authors aim to investigate the anti‐diabetic potential of genistein through IC50 value, fluorescence quenching and inhibition kinetics. Genistein was found to exhibit an inhibition activity on α‐glucosidase with an IC50 value of 7.79 ± 0.36 µm. Analysis of fluorescence spectra indicated that genistein quenched enzyme emission through a static mechanism with a bimolecular quenching constant, kq = 4.04 × 1013 m−1 s−1. In addition, it was found that genistein was bound to α‐glucosidase with a high affinity of binding constant, Ka = 3.38 × 105 m−1 and a 1:1 stoichiometric ratio (n = 1.06). In order to suggest a possible contact involved, 8‐anilino‐1‐naphthalenesulfonic acid (ANS) was added as an extrinsic fluorescence probe to enzyme solution and analyzed fluorescence spectra of the α‐glucosidase‐ANS complexes. When treated with genistein, fluorescence intensities of the complexes were reduced remarkably, indicating that genistein interacts with the enzyme via a hydrophobic domain. Finally, the inhibition constant and inhibition mode were studied by inhibition kinetics. The results revealed that genistein bound easily to α‐glucosidase with a Ki value of 25.61 × 10−6 m through a non‐competitive type.

Fluorometric Measurement of Calmodulin-Dependent Peptide-Protein Interactions Using Dansylated Calmodulin.

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.

Allosteric and substrate site effects of relevant amino acids on structural stability and flexibility of glutamate dehydrogenase-1 (GDH-1).

Mammalian GDH-1, which converts glutamate to alpha-ketoglutarate and ammonia while reducing NAD(P), is constitutively expressed in virtually all cells of the body. The extensive literature on GDH-1 suggests that functionally relevant GDH-1 structures may be distributed over a conformational flexibility landscape: polyhexamer, hexamer, trimer, monomer, monomer (unfolded), monomer (misfolded), polymer (disassembled). We have used both intrinsic (tryptophan) and extrinsic (ANS) fluorescent probes to study the dependence of the reversible hexamer to trimer dissociation on the concentrations of the denaturing agents urea and guanidine hydrochloride (GdnHCl). Dissociation at each denaturant concentration increases with increase in concentration and/or temperature, and these dependencies are shown to be affected by amino acids which bind to allosteric and/or substrate binding sites. Dissociation of the hexamer is accompanied by a large increase in the fluorescence of ANS due to binding to increase in exposed hydrophobic microdomains on the protein. In contrast, tryptophan fluorescence decreases due to increased exposure of tryptopan in the protein to the aqueous environment. Measurement were made of the influence of several known substrates and allosteric modifiers (i.e. a selection physiologically relevant monocarboxylic amino acids) on the GDH-1 hexamer to trimer transition. The observed effect of adding each amino acid is consistent with independent measurements of its binding affinity. From the temperature dependence of the fluorescence intensity changes for ANS we obtained activation parameters for the hexamer-trimer transitions. Our data are consistent with the hexamer/trimer transition in GDH-1 having significant regulatory function in vivo, perhaps contribution to/complementing allosteric regulation of the enzyme activity.

Recent advancements toward non-invasive imaging of retinal amyloid-beta for early detection of Alzheimer's disease.

Recent advancements toward non-invasive imaging of retinal amyloid-beta for early detection of Alzheimer's disease.

Candida albicans exhibit two classes of cell surface binding sites for serum albumin defined by their affinity, abundance and prospective role in interkingdom signalling.

Serum albumin binding to the yeast form of Candida albicans is described. Two populations of binding site are identified using two complementary spectroscopic techniques: an extrinsic fluorescent probe, 3-hexa-decanoyl-7-hydrocoumarin ([HEXCO) added to the C. albicans yeast cell surface that records the electrostatic surface potential and so responds to the surface binding of serum albumin and secondly a light scattering technique that reveals how albumin modulates aggregation of the yeast population. The albumin binding sites are found to possess different binding affinities and relative abundance leading to different total binding capacities. These are characterized as a receptor population with high affinity binding (Kd ~ 17 μM) but relatively low abundance and a separate population with high abundance but much lower affinity (Kd ~ 364 μM). The low-affinity binding sites are shown to be associated with the yeast cell aggregation. These values are found be dependent on the C. albicans strain and the nature of the culture media; some examples of these effects are explored. The possible physiological consequences of the presence of these sites are speculated in terms of evading the host’s immune response, biofilm formation and possible interkingdom signaling processes.

A hemicyanine based fluorescence turn-on sensor for amyloid fibril detection in the far-red region

The designing or identification of extrinsic fluorescence probes for the sensitive and selective detection of amyloid fibrils, which can emit in the red to near infrared region, is an extremely important area of research. Long wavelength emission offers various crucial advantages such as preventing auto-fluorescence and photo-damage of specimen, reduced scattering, large penetration depth, etc. Herein, we have identified a hemicyanine based organic dye molecule, LDS722, which displays an impressive fluorescence turn-on emission (~200 fold increase), in the near infrared region of the spectrum, in presence of insulin fibrils. LDS722 also displays a very high selectivity to bind to the fibrillar form of insulin as opposed to its native conformation. Remarkably, the probe also registers a very large Stokes' shift of ~170 nm and a huge red shift of ~120 nm in the excitation spectra. The significant shift in the absorbance maximum of the fibril-bound probe, from its initial free form, results in the visual colour change facilitating naked eye detection of the fibril positive solution. LDS722 could also efficiently monitor the kinetic progression of the fibrillation process without any interference. Importantly, LDS722 also shows a good performance in the human serum matrix with a LOD of 55 nM.

Amyloid-β oligomerization monitored by single-molecule stepwise photobleaching.

A major hallmark of Alzheimer's disease is the misfolding and aggregation of the amyloid- β peptide (Aβ). While early research pointed towards large fibrillar- and plaque-like aggregates as being the most toxic species, recent evidence now implicates small soluble Aβ oligomers as being orders of magnitude more harmful. Techniques capable of characterizing oligomer stoichiometry and assembly are thus critical for a deeper understanding of the earliest stages of neurodegeneration and for rationally testing next-generation oligomer inhibitors. While the fluorescence response of extrinsic fluorescent probes such as Thioflavin-T have become workhorse tools for characterizing large Aβ aggregates in solution, it is widely accepted that these methods suffer from many important drawbacks, including an insensitivity to oligomeric species. Here, we integrate several biophysics techniques to gain new insight into oligomer formation at the single-molecule level. We showcase single-molecule stepwise photobleaching of fluorescent dye molecules as a powerful method to bypass many of the traditional limitations, and provide a step-by-step guide to implementing the technique in vitro. By collecting fluorescence emission from single Aβ(1-42) peptides labelled at the N-terminal position with HiLyte Fluor 555 via wide-field total internal reflection fluorescence (TIRF) imaging, we demonstrate how to characterize the number of peptides per single immobile oligomer and reveal heterogeneity within sample populations. Importantly, fluorescence emerging from Aβ oligomers cannot be easily investigated using diffraction-limited optical microscopy tools. To assay oligomer activity, we also demonstrate the implementation of another biophysical method involving the ratiometric imaging of Fura-2-AM loaded cells which quantifies the rate of oligomer-induced dysregulation of intracellular Ca2+ homeostasis. We anticipate that the integrated single-molecule biophysics approaches highlighted here will develop further and in principle may be extended to the investigation of other protein aggregation systems under controlled experimental conditions.

8-Anilino-1-naphthalenesulfonate (ANS) as a probe for Poly(vinyl alcohol)(PVA) swelling

Poly (vinyl alcohol) (PVA) is a hydrophilic polymer developed for biomedical applications. Swelling properties of PVA gels is very important for its drug delivery applications. There are many ways to study the swelling behaviour. Here we are proposing a fluorescent study by using 8-anilino-1-naphthalenesulfonate (ANS) as an extrinsic fluorescent probe. When ANS incorporated PVA films are allowed to swell in water, there is a loss of fluorescence intensity at 453 nm and there is a new peak at 500 nm. Thus, ANS is proposed as a fluorescent probe for the sensing of hydration of PVA.

Study of Interaction between Extrinsic Fluorescence Probe and Dissolved Organic Matter using Density Functional Theory

Study of Interaction between Extrinsic Fluorescence Probe and Dissolved Organic Matter using Density Functional Theory

Equilibrium unfolding and refolding of black gram (Vignamungo) phaseolin

Equilibrium unfolding of black gram phaseolin (BGP) using guanidine hydrochloride and urea as denaturants, thermal unfolding, and refolding were studied using several spectroscopic probes and electrophoretic techniques. UV absorbance and intrinsic fluorescence revealed a three-stage unfolding process, while CD spectroscopy, extrinsic fluorescence probe ANS, and thermal denaturation followed a two-state unfolding transition. The β-sheet was more resistant to chemical denaturation than the α-helical regions of BGP. BGP and an analogous navy bean phaseolin (NBP) could revert back to their protomeric conformations from their partially unfolded states in 2 M denaturant concentrations, while only NBP could regain its native conformation from the completely unfolded state. BGP completely unfolded in urea and upon thermal denaturation could never regain its native structure. These differences in the refolding of two otherwise homologous proteins from their completely unfolded states were discussed in terms of why BGP is a preferred legume in mixed legume–cereal fermentations. Practical applications The major storage proteins of black gram and dry beans exhibited significant differences in their unfolding–refolding behavior. Unlike the dry bean phaseolin that refolds back to its native structure quite quickly, black gram phaseolin was able to maintain its unfolded state; thereby allowing the formation of soft, porous, and spongy network in several popular fermented and steamed products on the Indian subcontinent. This may explain the popularity of black gram in such mixed cereal–legume fermentations. Black gram is thus ideally suited for legume-fortified food products where such texture is desired. Furthermore, understanding of the folding–unfolding behavior of major storage proteins of legume will enhance our ability to genetically modify these proteins with sulfur-rich peptides to improve their nutritional quality by identifying the key structural motifs in these proteins that are essential and vital to the proper folding of the protein in situ in genetically modified seeds.

The porcine odorant-binding protein as molecular probe for benzene detection.

In recent years, air pollution has been a subject of great scientific and public interests for the strong impact on human health. Air pollution is due to the presence in the atmosphere of polluting substances, such as carbon monoxide, sulfur and nitrogen oxides, particulates and volatile organic compounds (VOCs), derived predominantly from various combustion processes. Benzene is a VOC belonging to group-I carcinogens with a toxicity widely demonstrated. The emission limit values and the daily exposure time to benzene (TLV-TWA) are 5μg/m3 (0.00157 ppm) and 1.6mg/m3 (0.5 ppm), respectively. Currently, expensive and time-consuming analytical methods are used for detection of benzene. These methods require to perform a few preliminary steps such as sampling, and matrices pre-treatments. In addition, it is also needed the support of specialized personnel. Recently, single-walled carbon nanotube (SWNTs) gas sensors with a limit detection (LOD) of 20 ppm were developed for benzene detection. Other innovative bioassay, called bio-report systems, were proposed. They use a whole cell (Pseudomona putida or Escherichia coli) as molecular recognition element and exhibit a LOD of about 10 μM. Here, we report on the design of a highly sensitive fluorescence assay for monitoring atmospheric level of benzene. For this purpose, we used as molecular recognition element the porcine odorant-binding protein (pOBP). 1-Aminoanthracene was selected as extrinsic fluorescence probe for designing a competitive fluorescence resonance energy transfer (FRET) assay for benzene detection. The detection limit of our assay was 3.9μg/m3, a value lower than the actual emission limit value of benzene as regulated by European law.

Interactions of Neuronal Calcium Sensor Dream with Zinc

Maria J. Santiago Estevez and Jaroslava Miksovska Department of Chemistry and Biochemistry, Florida International University Neuronal calcium sensors regulate several physiological processes in the brain and are linked to numerous pathological conditions such as autism, Parkinson and Alzheimer disease. Downstream regulatory element antagonist modulator (DREAM) is among the neuronal calcium sensor family, it is an EF-hand protein highly expressed in the central nervous system in various areas such as the hippocampus and the neural cortex. It is responsible for regulating the kinetics of potassium channels, gene expression, calcium homeostasis and enzymatic activity of presenilin. The characterization of the metal-binding properties of DREAM and how it affects DREAM structure and DREAM interactions with effector proteins is fundamental to understand its biological function. Essential metals like Mg2+, Zn2+ and Ca2+ play extremely important roles in biological processes through direct interactions with proteins. DREAM carries out two Ca2+ binding EF hands and one Mg2+ binding EF hand, however, DREAM binding to other biologically significant metals is not very well known. Interactions of DREAM with Zn2+ have been monitored by using intrinsic and extrinsic fluorescent probes. Data shows that Zn2+ associates to DREAM with Kd ∼ 200 μM. Zn2+ and it does not compete for the same binding site as Ca2+ as the changes in the protein tertiary structure are distinct from those observed upon Ca2+association. Considering increased concentration of Zn2+ in neuronal tissue (150 - 200 μM), these results point towards the potential role of Zn2+ in modulating DREAM interactions with other intracellular proteins.

Theoretical study of extrinsic fluorescence probe reacting with volatile organic compounds

Theoretical study of extrinsic fluorescence probe reacting with volatile organic compounds

In Vitro Protein Stability of Two Naturally Occurring Thiopurine S-Methyltransferase Variants: Biophysical Characterization of TPMT*6 and TPMT*8.

Thiopurine S-methyltransferase (TPMT) is a polymorphic enzyme involved in the metabolism and inactivation of thiopurine substances administered as immunosuppressants in the treatment of malignancies and autoimmune diseases. In this study, the naturally occurring variants, TPMT*6 (Y180F) and TPMT*8 (R215H), have been biophysically characterized. Despite being classified as low and intermediate in vivo enzyme activity variants, respectively, our results demonstrate a discrepancy because both TPMT*6 and TPMT*8 were found to exhibit normal functionality in vitro. While TPMT*8 exhibited biophysical properties almost indistinguishable from those of TPMTwt, the TPMT*6 variant was found to be destabilized. Furthermore, the contributions of the cofactor S-adenosylmethionine (SAM) to the thermodynamic stability of TPMT were investigated, but only a modest stabilizing effect was observed. Also presented herein is a new method for studies of the biophysical characteristics of TPMT and its variants using the extrinsic fluorescent probe 8-anilinonaphthalene-1-sulfonic acid (ANS). ANS was found to bind strongly to all investigated TPMT variants with a Kd of approximately 0.2 μM and a 1:1 binding ratio as determined by isothermal titration calorimetry (ITC). Circular dichroism and fluorescence measurements showed that ANS binds exclusively to the native state of TPMT, and binding to the active site was confirmed by molecular modeling and simulated docking as well as ITC measurements. The strong binding of the probe to native TPMT and the conformity of the obtained results demonstrate the advantages of using ANS binding characteristics in studies of this protein and its variants.

Chaotropes trigger conformational rearrangements differently in Concanavalin A

Concanavalin A (ConA) is a plant lectin having industrial and biological applications. Concanavalin A changes conformation upon exposure to different stress conditions, like exposure to sodium dodecyl sulphate, guanidine hydrochloride, varying hydronium ion potential, etc. The conformational changes were studied using circular dichroism spectroscopy and the structural rigidity of ConA was explored using fluorescence spectroscopy, taking tryptophan as an intrinsic and 8-anilino-1-naphthalenesulfonic acid as an extrinsic fluorescence probes, in different stress conditions. ConA loses the quaternary structure in all the studied stress conditions, which further leads to denaturation of the protein. However, the mechanism of denaturation varied with the studied conditions, like different SDS concentrations and hydronium ion potentials, wherein the protein undergoes a conformational rearrangement from $$\upbeta $$ -sheet to $$\upalpha $$ -helix. Moreover, GdnHCl triggered complete denaturation of ConA into a predominantly random coil conformation. The results suggested that denaturation of ConA follows different pathways depending on the chemical properties and concentrations of the denaturants used. SynopsisGuanidine hydrochloride shows complete unfolding of Concanavalin A (Con A) above 2 M concentration, whereas GdnHCl between 1–2 M triggered the molten globule-like conformation. At basic pH, ConA adopts all- $$\upbeta $$ -sheet conformation, and at acidic pH it retained demetallized monomeric jelly roll motif. Additionally, in the presence of SDS at >2 mM, ConA undergoes conformational rearrangement into a predominantly $$\upalpha $$ -helix conformation, rather a leticn-like all- $$\upbeta $$ -sheet conformation at lower concentrations of SDS.

Determination of the Critical Micelle Concentration of Triton X-100 Using the Compound 3-(benzoxazol-2-yl)-7-(N,N-diethylamino)chromen-2-one as Fluorescent Probe

In the present study we estimated the Critical Micelle Concentration (CMC) of Triton X-100 in an aqueous buffered medium (PBS buffer, pH 7.4), using 3-(benzoxazol-2-yl)-7-(N,N-diethylamino)chromen-2-one (BDC), a push-pull compound, as extrinsic fluorescence probe. The CMC value found, 0.33 mmol L -1 , shows good agreement with data from literature obtained using the fluorescence probe technique. Additionally, the polarity, in terms of the E T (30) scale, and the viscosity of the micelle microenvironment in which the probe is preferentially allocated, was estimated as being 46.9 kcal mol -1 and 70.3 cP, respectively, confirming that this microdomain is polar and highly viscous, with characteristics of polyethylene glycol groups consisting in the palisade layer at the micelle-water interface. DOI: http://dx.doi.org/10.17807/orbital.v0i0.923

Bile salt induced solubilization of methylene blue: Study on methylene blue fluorescence properties and molecular mechanics calculation

Methylene blue (MB) is a hydrophobic drug molecule, having importance both as a staining reagent and pharmaceutical agent. MB is strongly fluorescent, with an emission peak at 686nm (λex 665nm). In the study, the possibility of MB as an extrinsic fluorophore to study the micellization behavior of bile salts (BSs) was carried out. Since BSs are drug delivery systems, the solubilization of hydrophobic MB drug molecule by BSs was achieved and the nature of association of MB with BS media, namely sodium cholate (NaC) and sodium deoxycholate (NaDC) was evaluated. Change in the photophysical properties of MB is monitored through fluorescence intensity and fluorescence anisotropy at emission peak, 686nm of MB. Molecular mechanics calculations were carried out to evaluate the MB–BS association. The estimated heat of formation, ΔHf values are –625.19kcal/mol for MB–NaC and –757.48kcal/mol for MB–NaDC. The photophysical study also revealed that MB reports the step-wise aggregation pattern of BSs media, as an extrinsic fluorescence probe.

Dual-sensor fluorescent probes of surfactant-induced unfolding of human serum albumin.

Two extrinsic fluorescent probes, 3-(dimethylamino)-8,9,10,11-tetrahydro-7H-cyclohepta[a]naphthalen-7-one (1) and 7-(dimethylamino)-2,3-dihydrophenanthren-4(1H)-one (2), are used to probe the unfolding of human serum albumin by sodium dodecyl sulfate (SDS). These probes respond separately to the polarity and H-bond-donating ability of their surroundings. Competitive binding experiments show that fluorophore 1 binds to site I (domain IIA) and 2 binds to site II (domain IIIA). The local acidity of 1 in site I is out of the sensing range of 1, whereas the local acidity of 2 in site II is calculated to be nearly zero on Catalan's solvent acidity index. Both probes show that the first two equivalents of bound SDS result in a decrease in the local polarity of the binding sites. Each subsequent equivalent of SDS gives rise to a dramatic increase in polarity until HSA is saturated with seven molecules of SDS at the end of the specific binding domain. Compound 2 experiences an increase of acidity of 0.10 on Catalan's solvent acidity index through seven equivalents of SDS, but the local acidity for 1 is still out of range. The increase in acidity experienced by 2 is greater than the increase in polarity. This result is consistent with greater exposure of the carbonyl group in 2, but not the bulk of 2, to the aqueous solvent in site II of the SDS-saturated HSA complex.

PH-dependent conformational changes in the HCV NS3 protein modulate its ATPase and helicase activities.

The hepatitis C virus (HCV) infects 170 to 200 million people worldwide and is, therefore, a major health problem. The lack of efficient treatments that specifically target the viral proteins or RNA and its high chronicity rate make hepatitis C the cause of many deaths and hepatic transplants annually. The NS3 protein is considered an important target for the development of anti-HCV drugs because it is composed of two domains (a serine protease in the N-terminal portion and an RNA helicase/NTPase in the C-terminal portion), which are essential for viral replication and proliferation. We expressed and purified both the NS3 helicase domain (NS3hel) and the full-length NS3 protein (NS3FL) and characterized pH-dependent structural changes associated with the increase in their ATPase and helicase activities at acidic pH. Using intrinsic fluorescence experiments, we have observed that NS3hel was less stable at pH 6.4 than at pH 7.2. Moreover, binding curves using an extrinsic fluorescent probe (bis-ANS) and ATPase assays performed under different pH conditions demonstrated that the hydrophobic clefts of NS3 are significantly more exposed to the aqueous medium at acidic pH. Using fluorescence spectroscopy and anisotropy assays, we have also observed more protein interaction with DNA upon pH acidification, which suggests that the hydrophobic clefts exposure on NS3 might be related to a loss of stability that could lead it to adopt a more open conformation. This conformational change at acidic pH would stimulate both its ATPase and helicase activities, as well as its ability to bind DNA. Taken together, our results indicate that the NS3 protein adopts a more open conformation due to acidification from pH 7.2 to 6.4, resulting in a more active form at a pH that is found near Golgi-derived membranes. This increased activity could better allow NS3 to carry out its functions during HCV replication.