Tryptophan Residues Research Articles

Fluorine-19 labeling of the tryptophan residues in the G protein-coupled receptor NK1R using the 5-fluoroindole precursor in Pichia pastoris expression.

In NMR spectroscopy of biomolecular systems, the use of fluorine-19 probes benefits from a clean background and high sensitivity. Therefore, 19F-labeling procedures are of wide-spread interest. Here, we use 5-fluoroindole as a precursor for cost-effective residue-specific introduction of 5-fluorotryptophan (5F-Trp) into G protein-coupled receptors (GPCRs) expressed in Pichia pastoris. The method was successfully implemented with the neurokinin 1 receptor (NK1R). The 19F-NMR spectra of 5F-Trp-labeled NK1R showed one well-separated high field-shifted resonance, which was assigned by mutational studies to the "toggle switch tryptophan". Residue-selective labeling thus enables site-specific investigations of this functionally important residue. The method described here is inexpensive, requires minimal genetic manipulation and can be expected to be applicable for yeast expression of GPCRs at large.

Tryptophan residues in TDP-43 and SOD1 modulate the cross-seeding and toxicity of SOD1

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of motor neurons. Neuronal superoxide dismutase-1 (SOD1) inclusion bodies are characteristic of familial ALS with SOD1 mutations, while a hallmark of sporadic ALS is inclusions containing aggregated WT TAR DNA-binding protein 43 (TDP-43). We show here that co-expression of mutant or WT TDP-43 with SOD1 leads to misfolding of endogenous SOD1 and aggregation of SOD1 reporter protein SOD1G85R-GFP in human cell cultures and promotes synergistic axonopathy in zebrafish. Intriguingly, this pathological interaction is modulated by natively solvent-exposed tryptophans in SOD1 (tryptophan-32) and TDP-43 RNA-recognition motif RRM1 (tryptophan-172), in concert with natively sequestered TDP-43 N-terminal domain tryptophan-68. TDP-43 RRM1 intrabodies reduce WT SOD1 misfolding in human cell cultures, via blocking tryptophan-172. Tryptophan-68 becomes antibody-accessible in aggregated TDP-43 in sporadic ALS motor neurons and cell culture. 5-fluorouridine inhibits TDP-43-induced G85R-GFP SOD1 aggregation in human cell cultures and ameliorates axonopathy in zebrafish, via its interaction with SOD1 tryptophan-32. Collectively, our results establish a novel and potentially druggable tryptophan-mediated mechanism whereby two principal ALS disease effector proteins might directly interact in disease.

Fluorescence study of the interaction between albumin and layered double hydroxides

Layered double hydroxides nanoparticles (LDH-NP) are increasingly studied for biomedical applications. Nevertheless, their interaction with biomolecules such as proteins needs further exploration for an effective application. In this work, the adsorption of bovine serum albumin (BSA) on LDH-NP and the conformation changes of the protein upon adsorption were characterized using fluorescence spectroscopy. First, the quenching of tryptophan residues of BSA by chloride-intercalated LDH-NP was explored and the BSA adsorption capacity of LDH-NP were determined. Then, the structural conformation of the protein was analyzed by fluorescence spectroscopy (including synchronous, polarization and quenching studies) at different surface coverages. Finally, the proclivity of adsorbed BSA molecules to assemble as amyloid fibril was evaluated. Due to the positive charging and low curvature of LDH-NP, BSA molecules were strongly adsorbed, which produced a quenching of the protein fluorescence and a large adsorption capacity. The effect on BSA conformation was dependent on surface coverage (SC): at low values ,t he tryptophan residues were in more hydrophobic environments and more accessible to quenchers than al high ones. At low SC, there is space between the BSA molecules to spread on the surface, which led to a conformation change. Contrarily, the native conformation around tryptophan residues of BSA was preserved at high SC due to the tight packing of the adsorbed protein molecules. As a result, BSA molecules are stabilized against the formation of amyloid fibrils at high SC, while at low SC they present a similar fibrillation than free BSA.

Interaction of eugenol-based anti-tuberculosis nanoemulsion with bovine serum albumin: A spectroscopic study including Rifampicin, Isoniazid, Pyrazinamide, and Ethambutol

Tuberculosis (TB), a deadly infectious disease, is primarily caused by the bacterium Mycobacterium tuberculosis. The misuse of antibiotics has led to the development of drug resistance, prompting researchers to explore new technologies to combat multidrug-resistant Tuberculosis (MDR TB). Phospholipid-based nanotherapeutics, such as nanoemulsions, are gaining traction as they enhance drug solubility, stability, and bioavailability. Our study focuses on the interaction between Bovine Serum Albumin (BSA) and a drug-loaded nanoemulsion based on Eugenol. This nanoemulsion incorporates Eugenol, Clove, cinnamon oil, and first-line anti-tuberculosis drugs like Rifampicin, Isoniazid, Pyrazinamide, and Ethambutol. The primary objective is to assess the biosafety profile of the nanoemulsion upon interaction with BSA. We employed Fluorescence, UV–visible, and Fourier Transform Infrared Spectroscopy (FTIR) to analyze this interaction.UV–visible spectroscopy detected changes in hydrophobicity due to structural alterations in BSA near the tryptophan residue, leading to the formation of ground-state complexes. Fluorescence spectroscopy demonstrated that the nanoemulsion effectively quenched fluorescence originating from tryptophan and tyrosine residues. Studies using synchronous and three-dimensional spectroscopy point to a potential modification of the aromatic environment of BSA by the nanoemulsion. Resonance light scattering spectra indicated the formation of large aggregates due to the interaction with the nanoemulsion. The second derivative FTIR spectra showed an increase in the magnitude of secondary structure bands, suggesting a conformational shift. This research has significant pharmacological implications for developing safer, more targeted drug delivery systems. The information obtained from the interaction of the nanoemulsion with the blood carrier protein is vital for the future development of superior carriers with minimal adverse effects on patients. It is crucial to remember that conformational changes brought on by drug-ligand complexes attaching to carrier proteins may have negative consequences. Therefore, this study enhances the in vitro evaluation of potential adverse effects of the nanoemulsion on serum proteins.

Neopetromin, a Cyclic Tripeptide with a C-N Cross-Link, from the Marine Sponge Neopetrosia sp., That Causes Vacuole Fragmentation in Tobacco BY-2 Cells.

HPLC-MS analysis revealed the presence of an unreported peptide in the extract of the marine sponge Neopetrosia sp. Its structure was determined as a tripeptide, named neopetromin (1), composed of two tyrosine and one tryptophan residues with a heteroaromatic C-N cross-link between side chains. The absolute configuration of amino acids was determined using Marfey's method after ozonolysis and hydrolysis of 1. Compound 1 promoted vacuole fragmentation in an actin-independent manner in tobacco BY-2 cells.

Differential scanning fluorimetry to assess PFAS binding to bovine serum albumin protein.

The rapid screening of protein binding affinity for poly- and perfluoroalkyl substances (PFAS) benefits risk assessment and fate and transport modelling. PFAS are known to bioaccumulate in livestock through contaminated food and water. One excretion pathway is through milk, which may be facilitated by binding to milk proteins such as bovine serum albumin (BSA). We report a label-free differential scanning fluorimetry approach to determine PFAS-BSA binding over a broad temperature range. This method utilizes the tryptophan residue within the protein binding pocket as an intrinsic fluorophore, eliminating the need for fluorophore labels that may influence binding. BSA association constants were determined by (a) an equilibrium-based model at the melting temperature of BSA and (b) the Hill adsorption model to account for temperature dependent binding and binding cooperativity. Differences in binding between PFAS and fatty acid analogs revealed that a combination of size and hydrophobicity drives PFAS binding.

Modulation of sodium distribution and digestion properties in low salt myofibrillar protein gel incorporating γ-polyglutamic acid and transglutaminase: Conformation and microstructure

The weak gelling and poor digestion properties of myofibrillar protein (MP) under low-salt conditions constrain the development of functional sodium-reduced meat products. This study investigated how modification with γ-polyglutamic acid (γ-PGA) and transglutaminase (TG) cross-linking influences the conformation, gelation, and in vitro digestion of low-salt MPs pretreated with konjac glucomannan (KGM) and ultrasound. The results revealed that γ-PGA addition effectively facilitated the exposure of tryptophan and tyrosine residues in low salt MPs. The better dispersibility and slightly lower rheological properties of protein sols suggested the circumscribed effect of γ-PGA in augmenting protein gelation. TG crosslinking promoted moderate aggregation of MPs through different sites in myosin fragments, augmented the uniform distribution of sodium, and remarkably enhanced the gelling behavior of MPs. Importantly, the simultaneous incorporation of γ-PGA contributed to the refinement and uniformization of the TG cross-linked protein gel network, especially in the protein gel modified by pre-ultrasound treatment in combination with KGM. This result could also be corroborated by the augmented intermolecular interactions and enhanced digestibility observed in γ-PGA-modified TG crosslinked protein gel. Coupled with the identification of potentially bioactive peptides, it was confirmed that myosin light chain, actin, and myomesin account for the major contribution to the γ-PGA-induced changes in MP gels. Therefore, TG cross-linking combined with γ-PGA could be used to regulate the conformation and gelling properties of low-salt MPs, thus influencing the sodium distribution and digestion properties of low-salt meat products.

Studies on α-amylase inhibition by acarbose and quercetin using fluorescence, circular dichroism, docking, and dynamics simulations

This study looked at the effects of acarbose (ACA) and quercetin (QUE) on α-amylase activity, employing QUE and ACA to measure enzyme activity. The study observed that both drugs suppressed α-amylase activity, with greater inhibition reported at higher concentrations. The use of tryptophan residues as an intrinsic fluorescence probe permitted the observation of conformational changes in α-amylase, with CD measurements utilized to explore the secondary structure in the presence of QUE and ACA. Docking studies revealed an effective interaction between α-amylase, quercetin and acarbose, with a higher binding energy. Finally, a trajectory analysis was done to establish the stability and volatility of these complexes. These findings have potential significance for the development of new α-amylase-related therapeutics.

Site‐ and Stereoselective Glycomodification of Biomolecules through Carbohydrate‐Promoted Pictet–Spengler Reaction

AbstractCarbohydrates play pivotal roles in an array of essential biological processes and are consequently involved in many diseases. To meet the needs of glycobiology research, chemical enzymatic and non‐enzymatic methods have been developed to generate glycoconjugates with well‐defined structures. Herein, harnessing the unique properties of C6‐oxidized glycans, we report a straightforward and robust strategy for site‐ and stereoselective glycomodification of biomolecules with N‐terminal tryptophan residues by a carbohydrate‐promoted Pictet–Spengler reaction, which is not adapted to typical aldehyde substrates under biocompatible conditions. This method reliably delivers highly homogeneous glycoconjugates with stable linkages and thus has great potential for functional modulation of peptides and proteins in glycobiology research. Moreover, this reaction can be performed at the glycosites of glycopeptides, glycoproteins and living‐cell surfaces in a site‐specific manner. Control experiments indicated that the protected α‐O atom of aldehyde donors and free N−H bond of the tryptamine motif are crucial for this reaction. Mechanistic investigations demonstrated that the reaction exhibited a first‐order dependence on both tryptophan and glycan, and deprotonation/rearomatization of the pentahydro‐β‐carbolinium ion intermediate might be the rate‐determining step.

Site- and Stereoselective Glycomodification of Biomolecules through Carbohydrate-Promoted Pictet-Spengler Reaction.

Carbohydrates play pivotal roles in an array of essential biological processes and are consequently involved in many diseases. To meet the needs of glycobiology research, chemical enzymatic and non-enzymatic methods have been developed to generate glycoconjugates with well-defined structures. Herein, harnessing the unique properties of C6-oxidized glycans, we report a straightforward and robust strategy for site- and stereoselective glycomodification of biomolecules with N-terminal tryptophan residues by a carbohydrate-promoted Pictet-Spengler reaction, which is not adapted to typical aldehyde substrates under biocompatible conditions. This method reliably delivers highly homogeneous glycoconjugates with stable linkages and thus has great potential for functional modulation of peptides and proteins in glycobiology research. Moreover, this reaction can be performed at the glycosites of glycopeptides, glycoproteins and living-cell surfaces in a site-specific manner. Control experiments indicated that the protected α-O atom of aldehyde donors and free N-H bond of the tryptamine motif are crucial for this reaction. Mechanistic investigations demonstrated that the reaction exhibited a first-order dependence on both tryptophan and glycan, and deprotonation/rearomatization of the pentahydro-β-carbolinium ion intermediate might be the rate-determining step.

Bacillus cereus cereolysin O induces pyroptosis in an undecapeptide-dependent manner

Bacillus cereus is a clinically significant foodborne pathogen that causes severe gastrointestinal and non-gastrointestinal disease. Cereolysin O (CLO) is a putative virulence factor of B. cereus, and its function remains to be investigated. In this study, we examined the biological activity of CLO from a deep sea B. cereus isolate. CLO was highly toxic to mammalian cells and triggered pyroptosis through NLRP3 inflammasome-mediated caspase 1 and gasdermin D activation. CLO-induced cell death involved ROS accumulation and K+ efflux, and was blocked by serum lipids. CLO bound specifically to cholesterol, and this binding was essential to CLO cytotoxicity. The structural integrity of the three tryptophan residues in the C-terminal undecapeptide was vital for CLO to interact with membrane lipids and cause membrane perforation. Taken together, these results provided new insights into the molecular mechanism of B. cereus CLO-mediated cytotoxicity.

Oxidative cyclization reagents reveal tryptophan cation-π interactions.

Methods for selective covalent modification of amino acids on proteins can enable a diverse array of applications, spanning probes and modulators of protein function to proteomics1-3. Owing to their high nucleophilicity, cysteine and lysine residues are the most common points of attachment for protein bioconjugation chemistry through acid-base reactivity3,4. Here we report a redox-based strategy for bioconjugation of tryptophan, the rarest amino acid, using oxaziridine reagents that mimic oxidative cyclization reactions in indole-based alkaloid biosynthetic pathways to achieve highly efficient and specific tryptophan labelling. We establish the broad use of this method, termed tryptophan chemical ligation by cyclization (Trp-CLiC), for selectively appending payloads to tryptophan residues on peptides and proteins with reaction rates that rival traditional click reactions and enabling global profiling of hyper-reactive tryptophan sites across whole proteomes. Notably, these reagents reveal a systematic map of tryptophan residues thatparticipate in cation-π interactions, including functional sites that can regulate protein-mediated phase-separation processes.

A nanocomposite film with layer-by-layer self-assembled gold nanospheres driven by cucurbit[7]uril for the selective transport of l-tryptophan and lysozyme

Selective separation of amino acids and proteins is crucial in various areas of research, including proteomics, protein structure and function studies, protein purification and drug development, and biosensing and biodetection. A nanocomposite film is formed by combining layer-by-layer self-assembled gold nanospheres (AuNPs) driven by cucurbit[7]uril (CB[7]) and polymethyl methacrylate (PMMA) film. Due to the host-guest interactions, the selective transmission of l-tryptophan in the nanocomposite film is confirmed by the current-voltage measurements using a picoammeter. Furthermore, by adjusting the particle size of AuNPs to increase channel size, lysozyme containing multiple tryptophan residues can selectively pass through the nanocomposite film, indicating the high versatility and adaptability of the nanocomposite film. This study will provide a new direction for the selective separation of amino acids and proteins.

Biophysical and functional characterization of N-terminal domain of Human Interferon Regulatory Factor 6.

Interferon regulatory factor 6 (IRF6) has a key function in palate fusion during palatogenesis during embryonic development, and mutations in IRF6 cause orofacial clefting disorders. The in silico analysis of IRF6 is done to obtain leads for the domain boundaries and subsequently the sub-cloning of the N-terminal domain of IRF6 into the pGEX-2TK expression vector and successfully optimized the overexpression and purification of recombinant glutathione S-transferase-fused NTD-IRF6 protein under native conditions. After cleavage of the GST tag, NTD-IRF6 was subjected to protein folding studies employing Circular Dichroism and Intrinsic fluorescence spectroscopy at variable pH, temperature, and denaturant. CD studies showed predominantly alpha-helical content and the highest stability of NTD-IRF6 at pH 9.0. A comparison of native and renatured protein depicts loss in the secondary structural content. Intrinsic fluorescence and quenching studies have identified that tryptophan residues are majorly present in the buried areas of the protein and a small fraction was on or near the protein surface. Upon the protein unfolding with a higher concentration of denaturant urea, the peak of fluorescence intensity decreased and red shifted, confirming that tryptophan residues are majorly present in a more polar environment. While regulating IFNβ gene expression during viral infection, the N-terminal domain binds to the promoter region of Virus Response Element-Interferon beta (VRE-IFNβ). Along with the protein folding analysis, this study also aimed to identify the DNA-binding activity and determine the binding affinities of NTD-IRF6 with the VRE-IFNβ promoter region. The protein-DNA interaction is specific as demonstrated by gel retardation assay and the kinetics of molecular interactions as quantified by Biolayer Interferometry showed a strong affinity with an affinity constant (KD) value of 7.96 × 10-10 M. NTD-IRF6 consists of a mix of α-helix and β-sheets that show temperature-dependent cooperative unfolding between 40 °C and 55 °C. Urea-induced unfolding shows moderate tolerance to urea as the mid-transition concentration of urea (Cm) is 3.2 M. The tryptophan residues are majorly buried as depicted by fluorescence quenching studies. NTD-IRF6 has a specific and high affinity toward the promoter region of VRE-IFNβ.

Sensitivity enhancement of radical-pair magnetoreceptors as a result of spin decoherence.

Electron spin relaxation is, on many occasions, considered an elephant in the room that challenges the idea of a radical-pair compass, a leading hypothesis for the navigation of migratory avian species. It has been widely recognized that an effective radical-pair magnetoreceptor requires a relaxation time that is long enough for an external magnetic field as weak as the geomagnetic field to significantly modify the coherent spin dynamics. However, previous studies proposed that certain spin relaxation, far quicker than the radical recombination reactions, could enhance, rather than degrade, the directional sensitivity of a radical-pair magnetoreceptor. Here, I investigate relaxation effects on the singlet-triplet interconversion of a model radical pair and find that the enhancement effect originates from population relaxation over a period of several microseconds as a result of efficient spin decoherence. Insights into the truncated spin systems shed light on the physics behind them. I further investigate the possibilities of such enhancement in cryptochrome-based magnetoreception, in which electron hopping takes place between tryptophan residues.

Exploring the binding mechanisms of thermally and ultrasonically induced molten globule-like β-lactoglobulin with heptanal as revealed by multi-spectroscopic techniques and molecular simulation

This work employed the model protein β-lactoglobulin (BLG) to investigate the contribution of microstructural changes to regulating the interaction patterns between protein and flavor compounds through employing computer simulation and multi-spectroscopic techniques. The formation of molten globule (MG) state-like protein during the conformational evolution of BLG, in response to ultrasonic (UC) and heat (HT) treatments, was revealed through multi-spectroscopic characterization. Differential MG structures were distinguished by variations in surface hydrophobicity and the microenvironment of tryptophan residues. Fluorescence quenching measurements indicated that the formation of MG enhanced the binding affinity of heptanal to protein. LC-MS/MS and NMR revealed the covalent bonding between heptanal and BLG formed by Michael addition and Schiff-base reactions, and MG-like BLG exhibited fewer chemical shift residues. Molecular docking and molecular dynamics simulation confirmed the synergistic involvement of hydrophobic interactions and hydrogen bonds in shaping BLG-heptanal complexes thus promoting the stability of BLG structures. These findings indicated that the production of BLG-heptanal complexes was driven synergistically by non-covalent and covalent bonds, and their interaction processes were influenced by processes-induced formation of MG potentially tuning the release and retention behaviors of flavor compounds.

Supervised and unsupervised machine learning approaches for monitoring subvisible particles within an aluminum-salt adjuvanted vaccine formulation.

Suspensions of protein antigens adsorbed to aluminum-salt adjuvants are used in many vaccines and require mixing during vial filling operations to prevent sedimentation. However, the mixing of vaccine formulations may generate undesirable particles that are difficult to detect against the background of suspended adjuvant particles. We simulated the mixing of a suspension containing a protein antigen adsorbed to an aluminum-salt adjuvant using a recirculating peristaltic pump and used flow imaging microscopy to record images of particles within the pumped suspensions. Supervised convolutional neural networks (CNNs) were used to analyze the images and create "fingerprints" of particle morphology distributions, allowing detection of new particles generated during pumping. These results were compared to those obtained from an unsupervised machine learning algorithm relying on variational autoencoders (VAEs) that were also used to detect new particles generated during pumping. Analyses of images conducted by applying both supervised CNNs and VAEs found that rates of generation of new particles were higher in aluminum-salt adjuvant suspensions containing protein antigen than placebo suspensions containing only adjuvant. Finally, front-face fluorescence measurements of the vaccine suspensions indicated changes in solvent exposure of tryptophan residues in the protein that occurred concomitantly with new particle generation during pumping.

Identification and characterization of nuclear localization signals in the circumsporozoite protein of Plasmodium falciparum.

Secretory proteins of Plasmodium exhibit differential spatial and functional activity within the host cell nucleus. However, the nuclear localization signals (NLSs) for these proteins remain largely uncharacterized. In this study, we have identified and characterized two NLSs in the circumsporozoite protein of Plasmodium falciparum (Pf-CSP). Both NLSs in the Pf-CSP contain clusters of lysine and arginine residues essential for specific interactions with the conserved tryptophan and asparagine residues of importin-α, facilitating nuclear translocation of Pf-CSP. While the two NLSs of Pf-CSP function independently and are both crucial for nuclear localization, a single NLS of Pf-CSP leads to weak nuclear localization. These findings shed light on the mechanism of nuclear penetrability of secretory proteins of Plasmodium proteins.

Stabilization of a Cu-binding site by a highly conserved tryptophan residue

Copper serves as an essential cofactor for nearly all living organisms. There are still many gaps remaining in our knowledge of how Gram-positive bacteria import copper and maintain homeostasis. To obtain a better understanding of how these processes work, here we focus on the ycnKJI operon responsible for regulating copper levels in the Gram-positive bacterium Bacillus subtilis. This operon encodes three Cu-related proteins: a copper-dependent transcriptional repressor (YcnK), a putative copper importer (YcnJ), and a copper-binding protein of unknown function (YcnI). We previously found that YcnI's extracellular Domain of Unknown Function 1775 (DUF1775) houses a monohistidine brace motif that coordinates a single Cu(II) ion. The Cu(II) binding site includes a highly conserved tryptophan residue. Here, we investigate the role of that tryptophan and the ability of the protein to interact with other oxidation states of Cu. We find that YcnI exhibits strong preference for binding Cu in the oxidized Cu(II) state, and that the conserved tryptophan residue is not essential for the interaction. We determine the structure of a tryptophan variant to 1.95 Å resolution that indicates that the tryptophan is needed to stabilize the metal binding interaction, and find that this variant has weaker affinity for Cu(II) than the wild-type protein. Together, these data provide further insights into the DUF1775 domain family and reveal the role of the conserved tryptophan residue.

Multiple spectroscopic and computational studies on binding interaction of 2-phenylamino-4-phenoxyquinoline derivatives with bovine serum albumin

Binding characteristics of potent non-nucleoside HIV-1 reverse transcriptase inhibitors, 4-(2′,6′-dimethyl-4′-formylphenoxy)-2-(5″-cyanopyridin-2″ylamino) quinoline (1) and 4-(2′,6′-dimethyl-4′-cyanophenoxy)-2-(5″-cyanopyridin-2″ylamino) quinoline (2), to bovine serum albumin (BSA) under simulative physiological conditions were investigated by multiple spectroscopic and computational methods. The experimental results demonstrated that (1) and (2) bound to BSA at site III (subdomain IB), and quenched BSA fluorescence through a static quenching process. The binding interaction of (1) or (2) to BSA forms stable complexes with the binding constants (Kb) at the level of 104 L/mol and the number of binding site was determined to be 1 for both systems, indicating that new synthesized compounds occupied one site in BSA with moderate binding affinities. Based on the analysis of the thermodynamic parameters, it can be indicated that the main binding forces for interaction between BSA and both compounds were hydrogen bonding and van der Waals force. Synchronous fluorescence results revealed that the interaction of two compounds with BSA led to modifications in the microenvironment surrounding tryptophan residue of BSA. Circular dichroism spectra demonstrated alterations in the secondary structure of BSA induced by (1) and (2). Moreover, the experimental data of molecular docking and molecular dynamics (MD) simulations supported the results obtained from multiple spectroscopic techniques, confirming the binding interactions between both compounds and BSA.