Fluorescent Amino Acid Research Articles

Spectroscopic and molecular docking approach of the interaction of vitamins with human serum transferrin

The interaction of vitamins C, B12, and folic acid (B9) with human serum transferrin (HST) was studied. A static interaction mechanism for the binding of the vitamins with HST was revealed. The interaction takes place with small binding constants in the case of both vitamin C and B12, and a moderate constant in the case of folic acid. The driving forces of the interactions were: hydrophobic for vitamin B12, hydrogen bonding and van der Waals forces for vitamin C, and electrostatic forces for the folic acid. The microenvironments of its fluorescent amino acids, Trp and Tyr, were changed by all bound vitamins. The distances between HST and vitamins were determined, these data confirming the results obtained by fluorescence experiments. The denaturation temperature of HST was modified by the interactions with vitamins. Molecular docking simulation showed the best orientation of vitamins towards HST 3D structure and confirmed the fluorescence data.

Vanilloid agonist-mediated activation of TRPV1 channels requires coordinated movement of the S1–S4 bundle rather than a quiescent state

Transient receptor potential vanilloid1 (TRPV1) channel plays an important role in a wide range of physiological and pathological processes, and a comprehensive understanding of TRPV1 gating will create opportunities for therapeutic intervention. Recent incredible advances in cryo-electron microscopy (cryo-EM) have yielded high-resolution structures of all TRPV subtypes (TRPV1–6) and all of them share highly conserved six transmembrane (TM) domains (S1–S6). As revealed by the open structures of TRPV1 in the presence of a bound vanilloid agonist (capsaicin or resiniferatoxin), TM helicesS1 to S4 form a bundle that remains quiescent during channel activation, highlighting differences in the gating mechanism of TRPV1 and voltage-gated ion channels. Here, however, we argue that the structural dynamics rather than quiescence of S1–S4 domains is necessary for capsaicin-mediated activation of TRPV1. Using fluorescent unnatural amino acid (flUAA) incorporation and voltage-clamp fluorometry (VCF) analysis, we directly observed allostery of the S1–S4 bundle upon capsaicin binding. Covalent occupation of VCF-identified sites, single-channel recording, cell apoptosis analysis, and exploration of the role of PSFL828, a novel non-vanilloid agonist we identified, have collectively confirmed the essential role of this coordinated S1–S4 motility in capsaicin-mediated activation of TRPV1. This study concludes that, in contrast to cryo-EM structural studies, vanilloid agonists are also required for S1–S4 movement during TRPV1 activation. Redefining the gating process of vanilloid agonists and the discovery of new non-vanilloid agonists will allow the evaluation of new strategies aimed at the development of TRPV1 modulators.

Genetic Incorporation of Fluorescent Amino Acid into Fatty Acid Binding Protein for Fatty Acid Detection

Fatty acids play critical roles in biological processes, such as energy storage, metabolism, signal transduction, and immune regulation. Therefore, it is necessary to develop in-vitro fluorescent sensors to detect free fatty acids. By genetically incorporating a synthetic fluorescent amino acid (L-(7-hydroxycoumarin-4-yl) ethylglycine, Cou) into fatty acid-binding protein (FABP), we obtained a fluorescent sensor that has a turn-on signal in the presence of the fatty acids. Its response to medium-chain and long-chain fatty acids can be increased by 5.8-fold within several minutes, highlighting its potential applications in fatty acids-related biological processes. Our newly developed fatty acid detection system based on genetic expansion technology has extended the molecular toolboxes available for important biological molecular analysis.

Inhibitory activity and mechanism of guavinoside B from guava fruits against α-glucosidase: Insights by spectroscopy and molecular docking analyses.

Guavinoside B (GUB) is the main active substance in guava fruit and shows promising biological activities. In this study, the inhibitory activity and mechanism of GUB on α-glucosidase were studied by using spectroscopic techniques, kinetic analysis, and molecular docking. Results indicated that GUB possessed significant inhibition ability on α-glucosidase, which was about 10 times that of acarbose. The GUB was a mixed-type inhibitor, which suppressed the activity of α-glucosidase through a reversible process. Fluorescence analysis revealed that GUB quenched the fluorescence of α-glucosidase statically, the formation of GUB-α-glucosidase complex was a spontaneous and exothermic process, van der Waals forces, hydrogen bonding, and hydrophobic interaction were the predominant driving forces, only one single-binding site on α-glucosidase was involved in the binding process. GUB inserted into the hydrophobic pocket of α-glucosidase with 11 hydrogen bonds and two π-π stacking formed. The presence of GUB changed the microenvironment near the fluorescent amino acids of α-glucosidase, and the structure of α-glucosidase was slightly changed, eventually leading to the decrease of α-glucosidase activity. PRACTICAL APPLICATIONS: Diabetes mellitus (DM) is a worldwide chronic metabolic disease threatening human health seriously. Guava fruit is a popular fruit, and its extracts were reported to show many biological activities. GUB is the main benzophenone glycoside in guava fruits. However, the inhibitory activity and mechanism of its specific active compound GUB are still unclear. Studies have shown that GUB could reversibly inhibit the activity of α-glucosidase, and its inhibitory ability was about 10 times that of acarbose. The kinetics and mechanism of inhibition were revealed. These will facilitate the further research and application of guava fruit and GUB in functional and healthy foods against hyperglycinaemia or even DM.

Structural Basis for Blocked Excited State Proton Transfer in a Fluorescent, Photoacidic Non-Canonical Amino Acid-Containing Antibody Fragment

The fluorescent non-canonical amino acid (fNCAA) L-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) contains a photoacidic 7-hydroxycoumarin (7-HC) side chain whose fluorescence properties can be tuned by its environment. In proteins, many alterations to 7-HCAA’s fluorescence spectra have been reported including increases and decreases in intensity and red- and blue-shifted emission maxima. The ability to rationally design protein environments that alter 7-HCAA’s fluorescence properties in predictable ways could lead to novel protein-based sensors of biological function. However, these efforts are likely limited by a lack of structural characterization of 7-HCAA-containing proteins. Here, we report the steady-state spectroscopic and x-ray crystallographic characterization of a 7-HCAA-containing antibody fragment (in the apo and antigen-bound forms) in which a substantially blue-shifted 7-HCAA emission maximum (∼70 nm) is observed relative to the free amino acid. Our structural characterization of these proteins provides evidence that the blue shift is a consequence of the fact that excited state proton transfer (ESPT) from the 7-HC phenol has been almost completely blocked by interactions with the protein backbone. Furthermore, a direct interaction between a residue in the antigen and the fluorophore served to further block proton transfer relative to the apoprotein. The structural basis of the unprecedented blue shift in 7-HCAA emission reported here provides a framework for the development of new fluorescent protein-based sensors.

Genetically encoded fluorescent unnatural amino acids and FRET probes for detecting deubiquitinase activities.

Herein we present the genetic encoding of 7-aminocoumarin-based lysine derivatives, ACouK and AFCouK, into proteins in both bacterial and mammalian cells and the characterization of FRET pairs comprising ACouK or AFCouK as the donor and GFP as the acceptor. We further report the application of the FRET pairs to construct fully genetically encoded ratiometric probes for detecting deubiquitinases and screening for inhibitors.

Measuring Bulk Translation Activity in Single Mammalian Cells During the Integrated Stress Response.

The attenuation of global translation is a critical outcome of the integrated stress response (ISR). Consequently, it is important to effectively detect and measure protein synthesis in studies seeking to evaluate the ISR. This chapter details two methods, surface sensing of translation (SUnSET) and fluorescent noncanonical amino acid tagging (FUNCAT), to measure global translation activity in individual cells using fluorescence microscopy as a read-out. Detecting bulk translation activity in single cells is advantageous for the concurrent observation of newly synthesized proteins and other cellular structures and to identify differences in translation activity among individuals within a population of cells.

A Simplified Protocol to Incorporate the Fluorescent Unnatural Amino Acid ANAP into Xenopus laevis Oocyte-Expressed P2X7 Receptors.

The long intracellular P2X7 C-terminus accounts for diverse downstream effects of P2X7 activation. Although the recent determination of the cryo-EM structure of the full-length P2X7 receptor finally revealed the structure and several unexpected features of the large cytoplasmic domain, its molecular function remains enigmatic. Incorporation of unnatural amino acids (UAA) via an amber Stop codon has been a powerful tool for structure-function analysis of proteins. Voltage clamp fluorometry (VCF) with the fluorescent unnatural amino acid L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (ANAP) provides a means to study intracellular domain movements of ion channel receptors. In the Xenopus laevis oocyte expression system, site-specific introduction of this environment-sensitive fluorophore can be achieved by the nuclear injection of cDNA encoding an orthogonal amber suppressor tRNA/aminoacyl-tRNA synthetase pair and subsequent cytoplasmic injection of ANAP together with the respective cRNA containing the amber Stop codon. Here, we describe this protocol for expression of ANAP-labeled P2X7. In addition, we provide a simplified alternative protocol, in which we coinject cRNAs encoding the tRNA synthetase and mutant P2X7 together with the synthesized amber suppressor tRNA and ANAP in one step into the cytosol. We found that the new protocol yielded more reproducible results and was less harmful for the oocytes. By selective fluorescence labeling of the ANAP-labeled P2X7 protein in the oocyte plasma membrane and VCF recordings, we show that this method results in comparable levels of functional ANAP-labeled P2X7 protein.

Conformational dynamics during misincorporation and mismatch extension defined using a DNA polymerase with a fluorescent artificial amino acid

High-fidelity DNA polymerases select the correct nucleotide over the structurally similar incorrect nucleotides with extremely high specificity while maintaining fast rates of incorporation. Previous analysis revealed the conformational dynamics and complete kinetic pathway governing correct nucleotide incorporation using a high-fidelity DNA polymerase variant containing a fluorescent unnatural amino acid. Here we extend this analysis to investigate the kinetics of nucleotide misincorporation and mismatch extension. We report the specificity constants for all possible misincorporations and characterize the conformational dynamics of the enzyme during misincorporation and mismatch extension. We present free energy profiles based on the kinetic measurements and discuss the effect of different steps on specificity. During mismatch incorporation and subsequent extension with the correct nucleotide, the rates of the conformational change and chemistry are both greatly reduced. The nucleotide dissociation rate, however, increases to exceed the rate of chemistry. To investigate the structural basis for discrimination against mismatched nucleotides, we performed all atom molecular dynamics simulations on complexes with either the correct or mismatched nucleotide bound at the polymerase active site. The simulations suggest that the closed form of the enzyme with a mismatch bound is greatly destabilized due to weaker interactions with active site residues, nonideal base pairing, and a large increase in the distance from the 3ʹ-OH group of the primer strand to the α-phosphate of the incoming nucleotide, explaining the reduced rates of misincorporation. The observed kinetic and structural mechanisms governing nucleotide misincorporation reveal the general principles likely applicable to other high-fidelity DNA polymerases.

Real-Time Spatial and Temporal Analysis of the Translocation of the Apoptosis-Inducing Factor in Cells.

Translocation of the apoptosis-inducing factor (AIF) from the mitochondria to the nucleus is crucial for AIF-mediated apoptosis. However, the lack of methods for real-time spatial and temporal analysis of translocation of functional AIF is a large hurdle to gain a detailed understanding of this process. In this study, a genetic code expansion technique was developed to overcome this hurdle. Specifically, this technique was utilized to construct ANAP-AIF containing a small fluorescent amino acid (ANAP) at a specific site in cells. Additionally, we developed efficient fluorescence resonance energy-transfer systems consisting of ANAP-AIF and either yellow fluorescent protein (YFP)-fused cyclophilin A (CypA) or Hsp70, respective positive and negative regulators for AIF translocation to the nucleus. We found that apoptosis inducers, including apoptozole, 2-phenylethynesulfonamide (PES), myricetin, Bam7, reactivating p53 and inducing tumor apoptosis (RITA), brefeldin A, and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) promote translocation of mitochondrial AIF to the cytosol after 4 h incubation, reaching a maximum after 6-7 h. However, these substances did not enhance AIF translocation to the nucleus through the interaction of AIF with Hsp70 in the cytosol. On the other hand, treatment with apoptosis inducers, such as paclitaxel, silibinin, doxorubicin, actinomycin D, and camptothecin caused AIF translocation to the nucleus after 4 h incubation through AIF binding to CypA, reaching saturation after 6-7 h. It was also found that Hsp70 and CypA regulate AIF translocation in a mutually exclusive manner because they do not interact with AIF simultaneously in cells undergoing apoptosis. The results demonstrate clearly that ANAP-incorporated proteins are powerful to obtain a more in-depth understanding of protein translocation.

Arsenic Toxicity-Induced Physiological and Metabolic Changes in the Shoots of Pteris cretica and Spinacia oleracea.

Arsenic is a ubiquitous toxic element that can be accumulated into plant parts. The present study investigated the response of Pteris cretica and Spinacia oleracea to As treatment through the analysis of selected physiological and metabolic parameters. Plants were grown in pots in As(V) spiked soil (20 and 100 mg/kg). Plants’ physiological condition was estimated through the determination of elements, gas-exchange parameters, chlorophyll fluorescence, water potential, photosynthetic pigments, and free amino acid content. The results confirmed differing As accumulation in plants, as well as in shoots and roots, which indicated that P. cretica is an As-hyperaccumulator and that S. oleracea is an As-root excluder. Variations in physiological and metabolic parameters were observed among As treatments. Overall, the results revealed a significant effect of 100 mg/kg As treatment on the analysed parameters. In both plants, this treatment affected growth, N, Mg, S, Mn, and Zn content, as well as net photosynthetic rate, chlorophyll fluorescence, and total free amino acid content. In conclusion, the results reflect the similarity between P. cretica and S. oleracea in some aspects of plants’ response to As treatment, while physiological and metabolic parameter changes related to As treatments indicate the higher sensitivity of S. oleracea.

Fast label-free identification of bacteria by synchronous fluorescence of amino acids.

Fast identification of pathogenic bacteria is an essential need for patient's diagnostic in hospitals and environmental monitoring of water and air quality. Bacterial cells consist of a very high amount of biological molecules whose content changes in response to different environmental conditions. The similarity between the molecular compositions of different bacterial cells limits the possibility to find unique markers to enable differentiation among species. Although many biological molecules in the cells absorb at the UV-Vis region, only a few of them can be detected in whole cells by their intrinsic fluorescence. Among these molecules are the amino acids phenylalanine, tyrosine, and tryptophan. In this work, we develop a rapid method for bacterial identification by synchronous fluorescence. We show that we can quantify the concentration for the 3 amino acids without any significant interference from other fluorophores in the cells and that we can differentiate among 6 pathogenic bacterial species by using the concentrations of their amino acids as a bacterial fingerprint. Fluorescent amino acids exist in all living cells. Therefore, this method has the potential to be applicative for the rapid identification of cells from all kinds of organisms.

Spectral and time-resolved photoluminescence of human platelets doped with platinum nanoparticles.

This paper describes a detailed study of spectral and time-resolved photoprocesses in human platelets and their complexes with platinum (Pt) nanoparticles (NPs). Fluorescence, quantum yield, and platelet amino acid lifetime changes in the presence and without femtosecond ablated platinum NPs have been studied. Fluorescence spectroscopy analysis of main fluorescent amino acids and their residues (tyrosine (Tyr), tryptophan (Trp), and phenylalanine (Phe)) belonging to the platelet membrane have been performed. The possibility of energy transfer between Pt NPs and the platelet membrane has been revealed. Förster Resonance Energy Transfer (FRET) model was used to perform the quantitative evaluation of energy transfer parameters. The prospects of Pt NPs usage deals with quenching-based sensing for pathology’s based on platelet conformations as cardiovascular diseases have been demonstrated.

Excitation, emission, and synchronous fluorescence for astrochemical applications: Experiments and computer simulations of synchronous spectra of polycyclic aromatic hydrocarbons and their mixtures

Synchronous fluorescence spectroscopy is a variation of the fluorescence technique for detection and characterization of many single ring aromatic organic molecules, polycyclic aromatic hydrocarbons, and fluorescent amino acids among other molecules. To our knowledge this technique has not been used in space exploration. In order to demonstrate the utility of this technique for planetary and astrochemical applications, we present several laboratory experiments and computer simulations with pure samples and mixtures. Computer deconvolution of experimental excitation and emission fluorescence bands is presented and used to generate synchronous spectra. The computer simulation successfully predicts the number of synchronous fluorescence (SF) bands, band shapes, and band maximum wavelengths for any constant wavelength difference (Δλ). To test the simulation, emission, excitation, and synchronous spectra were obtained for anthracene in n-hexane. Excellent agreement is obtained reproducing and finding the origin of the experimental SF bands for values of Δλ between 2 and 100. The excitation, emission, and synchronous (Δλ = 10) spectra of toluene, aniline, naphthalene, acenaphthene, pyrene, and anthracene are obtained. Excitation of a mixture of the six compunds at three wavelengths (249, 266, and 355 nm) produce fluorescence emission spectra identifying some compounds. The synchronous spectrum (Δλ = 10) of the same mixture is presented and assigned based on the synchronous bands of the individual compounds. The synchronous fluorescence technique and the computer simulation method are proposed to complement other techniques in the analysis of fluorescent samples from comets, as well as in missions to planets and satellites of the solar system.

Structural Origins of Altered Spectroscopic Properties upon Ligand Binding in Proteins Containing a Fluorescent Noncanonical Amino Acid.

Fluorescent noncanonical amino acids (fNCAAs) could serve as starting points for the rational design of protein-based fluorescent sensors of biological activity. However, efforts toward this goal are likely hampered by a lack of atomic-level characterization of fNCAAs within proteins. Here, we describe the spectroscopic and structural characterization of five streptavidin mutants that contain the fNCAA l-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) at sites proximal to the binding site of its substrate, biotin. Many of the mutants exhibited altered fluorescence spectra in response to biotin binding, which included both increases and decreases in fluorescence intensity as well as red- or blue-shifted emission maxima. Structural data were also obtained for three of the five mutants. The crystal structures shed light on interactions between 7-HCAA and functional groups, contributed either by the protein or by the substrate, that may be responsible for the observed changes in the 7-HCAA spectra. These data could be used in future studies aimed at the rational design of fluorescent, protein-based sensors of small molecule binding or dissociation.

Enantioselective Synthesis and Application of Small and Environmentally Sensitive Fluorescent Amino Acids for Probing Biological Interactions.

Environmentally sensitive fluorescent amino acids (FlAAs) have been used extensively to probe biological interactions. However, most of these amino acids are large and do not resemble amino acid side chains. Here, we report the enantioselective synthesis of two small and environmentally sensitive fluorescent amino acids bearing 7-dialkylaminocoumarin side chains by alkylation of a Ni(II) glycine Schiff base complex. These amino acids exhibit a large increase in fluorescence as environment polarity decreases. One of these FLAAs was incorporated into a highly active analog of the cyclic lipopeptide antibiotic paenibacterin by Fmoc solid-phase peptide synthesis via a new and very efficient route. This peptide was used to probe the interaction of the antibiotic with model liposomes, lipopolysaccharides, and live bacteria.

A Study of the Interaction, Morphology, and Structure in Trypsin-Epigallocatechin-3-Gallate Complexes.

Understanding the interaction between proteins and polyphenols is of significance to food industries. The aim of this research was to investigate the mode of aggregation for trypsin-EGCG (Epigallocatechin-3-gallate) complexes. For this, the complex was characterized by fluorescence spectroscopy, circular dichroism (CD) spectra, small-angel X-ray scattering (SAXS), and atomic force microscope (AFM) techniques. The results showed that the fluorescence intensity of trypsin-EGCG complexes decreased with increasing the concentration of EGCG, indicating that the interaction between trypsin and EGCG resulted in changes in the microenvironment around fluorescent amino acid residues. The results of CD analysis showed conformational changes in trypsin after binding with EGCG. The results from SAXS analysis showed that the addition of EGCG results in the formation of aggregates of trypsin-EGCG complexes, and increasing the concentration of EGCG resulted in larger aggregates. AFM images showed that the trypsin-EGCG complex formed aggregates of irregular ellipsoidal shapes with the size of about 200 × 400 × 200 nm, with EGCG interconnecting the trypsin particles. Overall, according to these results, it was concluded that the large aggregates of trypsin-EGCG complexes are formed from several small aggregates that are interconnected. The results of this study shed some light on the interaction between digestive enzymes and EGCG.

Fluorescent Amino Acid Initiated de novo Cyclic Peptides for the Label-Free Assessment of Cell Permeability*.

The major obstacle in applying peptides to intracellular targets is their low inherent cell permeability. Standard approaches to attach a fluorophore (e. g. FITC, TAMRA) can change the physicochemical properties of the parent peptide and influence their ability to penetrate and localize in cells. We report a label‐free strategy for evaluating the cell permeability of cyclic peptide leads. Fluorescent tryptophan analogues 4‐cyanotryptophan (4CNW) and β‐(1‐azulenyl)‐L‐alanine (AzAla) were incorporated into in vitro translated macrocyclic peptides by initiator reprogramming. We then demonstrate these efficient blue fluorescent emitters are good tools for monitoring peptide penetration into cells.

Synthesis of molecularly imprinted polymers using an amidine-functionalized initiator for carboxylic acid recognition

In traditional molecular imprinting reactions, the initial radicals generated by thermo- or photo-decomposition are randomly distributed in the reaction mixture. Because a noticeable portion of the initial radicals is able to cause self-polymerization of the crosslinking monomer, a significant part of the polymer product does not contain successfully imprinted molecular recognition sites. To solve this problem, we designed a molecular imprinting method using functionalized radical initiator to replace the conventional combination of initiator and functional monomer. Since the active radicals in the reaction mixture carry a template-binding moiety, the actual radical polymerization becomes more likely to take place nearby the molecular template. As a result, the efficiency of molecular imprinting can be improved. In this work, we report the use of amidine-functionalized initiator to synthesize molecularly imprinted polymers (MIPs) for selective recognition of methotrexate, a cytostatic drug used for cancer therapy. As glutamic acid represents a substructure of methotrexate, we select to use N-terminal protected glutamic acid as template to synthesize the MIPs. An initiator, 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) is used to provide strong interaction with the molecular template. Two MIPs synthesized using glutamic acid derivatives as templates display specific binding to the fluorescent amino acid derivative Fmoc-Glu. When the molecular binding is tested against methotrexate, the MIP particles also exhibit specific binding for the cytostatic drug. Using cationic functional initiator to target carboxyl epitope of molecular target, this work provides an additional example of molecular imprinting based on functionalized radical initiators.

In Vivo Behavior of the Antibacterial Peptide Cyclo[RRRWFW], Explored Using a 3-Hydroxychromone-Derived Fluorescent Amino Acid.

Labeling biomolecules with fluorescent labels is an established tool for structural, biochemical, and biophysical studies; however, it remains underused for small peptides. In this work, an amino acid bearing a 3-hydroxychromone fluorophore, 2-amino-3-(2-(furan-2-yl)-3-hydroxy-4-oxo-4H-chromen-6-yl)propanoic acid (FHC), was incorporated in a known hexameric antimicrobial peptide, cyclo[RRRWFW] (cWFW), in place of aromatic residues. Circular dichroism spectropolarimetry and antibacterial activity measurements demonstrated that the FHC residue perturbs the peptide structure depending on labeling position but does not modify the activity of cWFW significantly. FHC thus can be considered an adequate label for studies of the parent peptide. Several analytical and imaging techniques were used to establish the activity of the obtained labeled cWFW analogues toward animal cells and to study the behavior of the peptides in a multicellular organism. The 3-hydroxychromone fluorophore can undergo excited-state intramolecular proton transfer (ESIPT), resulting in double-band emission from its two tautomeric forms. This feature allowed us to get insights into conformational equilibria of the labeled peptides, localize the cWFW analogues in human cells (HeLa and HEK293) and zebrafish embryos, and assess the polarity of the local environment around the label by confocal fluorescence microscopy. We found that the labeled peptides efficiently penetrated cancerous cells and localized mainly in lipid-containing and/or other nonpolar subcellular compartments. In the zebrafish embryo, the peptides remained in the bloodstream upon injection into the cardinal vein, presumably adhering to lipoproteins and/or microvesicles. They did not diffuse into any tissue to a significant extent during the first 3 h after administration. This study demonstrated the utility of fluorescent labeling by double-emission labels to evaluate biologically active peptides as potential drug candidates in vivo.