Tryptophan Residues In Proteins Research Articles

Electrochemical Bioconjugation of Tryptophan Residues: A Strategy for Peptide Modification.

Interest in electrocatalytic bioconjugation reactions has surged, particularly for modifying tryptophan and tyrosine residues in proteins. We used a cost-effective graphite felt electrode and low-current methodology to achieve selective bioconjugation of tryptophan with thiophenols, yielding up to 92%. This method exclusively labeled tryptophan residues and incorporated fluorinated tryptophan for NMR analysis. Eight polypeptides, including lanreotide and leuprorelin, were effectively coupled, demonstrating the method's versatility and potential for novel diagnostic and therapeutic agents.

Photoredox C(2)-Arylation of Indole- and Tryptophan-Containing Biomolecules.

We introduce a novel and straightforward methodology for photoredox arylation of an indole scaffold using aryldiazonium salts under mild and metal-free conditions. Our approach enables the regioselective and chemoselective introduction of several aryl groups to the C(2) position of indoles and tryptophan, even in competition with other amino acids. This approach extends to the late-stage functionalization of peptides and lysozyme, heralding the unprecedented arylation of tryptophan residues in wild-type proteins and offering broad utility in chemical biology.

Investigating the biophysical interaction of serum albumins-gold nanorods using hybrid spectroscopic and computational approaches with the intent of enhancing cytotoxicity efficiency of targeted drug delivery

In this contribution, biophysical aspects of binding interaction, conformational changes, and molecular simulation studies of human and bovine serum albumins (HSA and BSA) exposed to gold nanorods (AuNRs) are thoroughly investigated and their bio-nanoconjugates were then used in in-vitro cytotoxic studies. The structural variations of AuNRs in the presence of serum proteins were confirmed by absorbance and high-resolution transmission electron microscopic (HR-TEM) pictures. According to the results of the steady-state emission quenching, AuNRs affect the intrinsic emission of HSA and BSA by a static mechanism. Synchronous emission spectrum results showed that the microenvironment near the tryptophan and tyrosine amino acid residues of serum proteins is altered, and the impact is stronger toward tryptophan than tyrosine. Furthermore, it was demonstrated by infrared, absorbance, and three-dimensional (3D) luminescence spectroscopic methods that the binding of AuNRs can arouse concerns and cause changes in the structural and/or microenvironmental characteristics of HSA and BSA. An outcome of the computational molecular simulation study supports the stability of the bio-nanoconjugate, and molecular docking interaction investigations suggest that the two-dimensional AuNRs surface associated with the cysteine residue of HSA and BSA contains sulphur atoms. Furthermore, using an in vitro model using the A-549 and MCF-7 cell lines, we calculated the toxicity and the effectiveness of cellular absorption of the AuNRs and their bio-nanoconjugates as a function of supplied dosage. These cytotoxicity findings demonstrate that protein adsorption affects AuNRs' physicochemical characteristics as well as enhances their subsequent biological function.

Interaction mechanism of icariin and whey protein based on spectrofluorimetry and molecular docking

Icariin has low bioavailability and poor stability, which limits its wide application. The complexation of icariin and whey protein is expected to solve this problem, but there is no research on their interaction mechanism. In view of this, the related mechanism was studied systematically by spectrofluorimetry and molecular docking method in this study. The fluorescence analysis showed that icariin and whey protein could form a non-covalent complex driven by hydrophobic force, which led to the fluorescence quenching of whey protein. In this process, the microenvironment around the tyrosine residue and tryptophan residue of whey protein changed. The molecular docking analysis confirmed the existence of hydrophobic interaction and hydrogen bonding in the complex, which well confirmed fluorescence results. The obtained results can promote the application of icariin in food.

Изменения свойств эритроцитов крови у родительниц после кесарева сечения в зависимости от метода ведения периоперационного периода

Aim: To study the changes in the structural and functional parameters of blood erythrocytes in maternity women during cesarean section, depending on the method of management of the perioperative period (PP), at all its stages. Design: Comparative group retrospective and prospective study. Materials and methods. The study included patients (n = 81) who underwent a planned cesarean section under spinal anesthesia (SA). The control group consisted of 38 maternity hospitals, in which PP was conducted traditionally. In the main group, 43 maternity hospitals were conducted according to the accelerated recovery program: refusal of mechanical intestinal cleansing, glucose-containing drink 2 hours before surgery, antibiotic prophylaxis 1 hour before surgery (cefazolin 2 g intravenously). Blood sampling and examination of the structural and dynamic parameters of the erythrocyte membranes of the patients' blood were carried out at different stages of PP: before CA, after the development of CA, by the end of the operation; umbilical cord blood was also examined. Results. In the control group, the polarity of annular and total lipids, as well as the microviscosity of total lipids, did not significantly change at different stages of Cesarean section. By the end of the operation, the fluidity of annular lipids increased by 24% and the parameter of the efficiency of the transfer of excitation energy from tryptophan residues of erythrocyte membrane proteins by umbilical cord blood to pyrene ((F0–F)/F0) increased by 11%. In the main group, after the development of CA and by the end of the operation, the index (F0–F)/F0 in the membranes of red blood cells decreased by 10%; the fluidity of annular lipids increased to CA and by the end of the operation by 25%, after the development of CA — by 30% relative to the control group to CA. The dissociation constants of 1-anilinonaphthalene-8-sulfonate with plasma proteins and erythrocyte membranes in the control group up to CA differ significantly: Kd2 is 5.34 times greater than Kd1. The number of binding centers in erythrocytes is 12 times less than in plasma proteins. Conclusion. The concept of PUV, which promotes rapid recovery of the patient, can prevent the intensification of oxidative-inflammatory processes, which allows the development of new therapeutic methods to improve the rheological properties of blood in many clinical conditions. Keywords: perioperative period, accelerated recovery program, carbohydrate drink, caesarean section, conformational changes in proteins.

Characterization of the binding between anti-tumor drug 5-fluorouracil and human alpha-2-macroglobulin: spectroscopic and molecular docking analyses

5-Fluorouracil (5-FU) is a well-recognized anticancer drug used in the treatment of tumors of head, neck and breast. Drug pharmacokinetics is affected upon binding with protein, thus, making drug–protein interactions imperative to study. Present work investigates the interaction between 5-FU and human major antiproteinase-alpha-2-macroglobulin (α2M) by multi-spectroscopic, calorimetric and molecular docking techniques. UV/Visible absorption, intrinsic fluorescence and circular dichroism (CD) spectroscopic methods have been employed to unveil the mode and mechanism of 5-FU-α2M interaction. Synchronous fluorescence showed alteration in the microenvironment of tryptophan and tyrosine residues of protein. Far UV-CD spectra suggest slight alterations in the secondary structure of α2M by 5-FU. Thermodynamic parameters determined by fluorescence quenching experiments and isothermal titration calorimetry (ITC) suggested the involvement of hydrogen bonds and hydrophobic interactions. Moreover, ITC corroborate the spontaneous and exothermic nature of the interaction process. Molecular docking illustrates that 5-FU binds with moderate affinity and Asp953, Tyr1264, Lys1236, Thr1232, Tyr1323 and Leu951 were the main residues involved. Molecular dynamics simulation studies suggested that 5-FU was stabilizing the α2M structure and forming a stable complex. It was concluded that 5-FU lower the antiproteolytic activity of α2M significantly and causes disruption in the native structure and conformation of α2M. Communicated by Ramaswamy H. Sarma

Cross-linking and modification of fibronectin by peroxynitrous acid: Mapping and quantification of damage provides a new model for domain interactions

Fibronectin (FN) is an abundant glycoprotein found in plasma and the extracellular matrix (ECM). It is present at high concentrations at sites of tissue damage, where it is exposed to oxidants generated by activated leukocytes, including peroxynitrous acid (ONOOH) formed from nitric oxide (from inducible nitric oxide synthase) and superoxide radicals (from NADPH oxidases and other sources). ONOOH reacts rapidly with the abundant tyrosine and tryptophan residues in ECM proteins, resulting in the formation of 3-nitrotyrosine, di-tyrosine, and 6-nitrotryptophan. We have shown previously that human plasma FN is readily modified by ONOOH, but the extent and location of modifications, and the role of FN structure (compact versus extended) in determining these factors is poorly understood. Here, we provide a detailed LC-MS analysis of ONOOH-induced FN modifications, including the extent of their formation and the sites of intramolecular and intermolecular cross-links, including Tyr-Tyr, Trp-Trp, and Tyr-Trp linkages. The localization of these cross-links to specific domains provides novel data on the interactions between different modules in the compact conformation of plasma FN and allows us to propose a model of its unknown quaternary structure. Interestingly, the pattern of modifications is significantly different to that generated by another inflammatory oxidant, HOCl, in both extent and sites. The characterization and quantification of these modifications offers the possibility of the use of these materials as specific biomarkers of ECM modification and turnover in the many pathologies associated with inflammation-associated fibrosis.

Chemical modifications of tryptophan residues in peptides and proteins.

Chemical protein modifications facilitate the investigation of natural posttranslational protein modifications and allow the design of proteins with new functions. Proteins can be modified at a late stage on amino acid side chains by chemical methods. The indole moiety of tryptophan residues is an emerging target of such chemical modification strategies because of its unique reactivity and low abundance. This review provides an overview of the recently developed methods of tryptophan modification at the peptide and protein levels.

Analysis of the glycosylation products of peanut protein and lactose by cold plasma treatment: Solubility and structural characteristics

Cold plasma (CP) treatment was used to prepare the glycosylation conjugates of high-temperature peanut protein isolate (HPPI) and lactose to improve the solubility of HPPI. We observed that by increasing the CP treatment time to 3 min, the solubility of the conjugates increased to 1.34 mg/mL. An increase in the degree of glycosylation and a decrease in the degree of browning indicated that although CP treatment accelerated the glycosylation of HPPI and lactose, it interfered with the formation of melanoidin. The analysis of protein tertiary structure showed that tryptophan and tyrosine residues in proteins undergoing CP treatment were the primary sites for the Maillard reaction. The relative decrease in surface hydrophobicity and FT-IR analysis indicated that the increase in the -OH stretching vibration intensity on the protein surface represented the formation of the covalent bonds between HPPI and lactose during the CP treatment. An increase in the denaturation temperature of proteins was observed after grafting with lactose. Changes in the secondary structure and surface structure of proteins showed that lactose covalently bonded to the surface of HPPI during CP treatment, forming a more stable ordered structure.

Selective Modification of Tryptophan Residues in Peptides and Proteins Using a Biomimetic Electron Transfer Process.

We report here a photochemical process for the selective modification of tryptophan (Trp) residues in peptides and small proteins using electron-responsive N-carbamoylpyridinium salts and UV-B light. Preliminary mechanistic experiments suggest that the photoconjugation process proceeds through photoinduced electron transfer (PET) between Trp and the pyridinium salt, followed by fragmentation of the pyridinium N-N bond and concomitant transfer of this group to Trp. The reaction displays excellent site selectivity for Trp and is tolerant to other, redox-active amino-acid residues. Moreover, the reaction proceeds in pure aqueous conditions without the requirement of organic cosolvents or photocatalysts, is enhanced by glutathione, and operates efficiently over a wide range of peptide concentrations (10-700 μM). The scope of the process was explored through the labeling of 6-Trp-containing peptides and proteins ranging from 1 to 14 kDa. We demonstrate the versatility of the N-carbamoylpyridinium salt both by tuning the electrochemical and photochemical properties of the pyridinium scaffold to enable challenging photoconjugation reactions and by using the carbamoyl moiety to tether a plethora of productive functional groups, including reactive handles, purification tags, and removable protecting groups.

Exploration of reaction rates of chlorine dioxide with tryptophan residue in oligopeptides and proteins

Chlorine dioxide (ClO2), an alternative disinfectant to chlorine, has a superior ability to inactivate microorganisms, in which protein damage has been considered as the main inactivation mechanism. However, the reactivity of ClO2 with amino acid residues in oligopeptides and proteins remains poorly investigated. In this research, we studied the reaction rate constants of ClO2 with tryptophan residues in five heptapeptides and four proteins using stopped-flow or competition kinetic method. Each heptapeptide and protein contain only one tryptophan residue and the reactivity of tryptophan residue with ClO2 was lower than that of free tryptophan (3.88 × 104 (mol/L)−1sec−1 at pH 7.0). The neighboring amino acid residues affected the reaction rates through promoting inter-peptide aggregation, changing electron density, shifting pKa values or inducing electron transfer via redox reactions. A single amino acid residue difference in oligopeptides can make the reaction rate constants differ by over 60% (e.g. 3.01 × 104 (mol/L)−1sec−1 for DDDWNDD and 1.85 × 104 (mol/L)−1sec−1 for DDDWDDD at pH 7.0 (D: aspartic acid, W: tryptophan, N: asparagine)). The reaction rates of tryptophan-containing oligopeptides were also highly pH-dependent with higher reactivity for deprotonated tryptophan than the neutral specie. Tryptophan residues in proteins spanned a 4-fold range reactivity toward ClO2 (i.e. 0.84 × 104 (mol/L)−1sec−1 for ribonuclease T1 and 3.21 × 104 (mol/L)−1sec−1 for melittin at pH 7.0) with accessibility to the oxidant as the determinating factor. The local environment surrounding the tryptophan residue in proteins can also accelerate the reaction rates by increasing the electron density of the indole ring of tryptophan or inhibit the reaction rates by inducing electron transfer reactions. The results are of significance in advancing understanding of ClO2 oxidative reactions with proteins and microbial inactivation mechanisms.

How to evaluate the cellular uptake of CPPs with fluorescence techniques: Dissecting methodological pitfalls associated to tryptophan-rich peptides

Cell-penetrating peptides (CPP) are broadly recognized as efficient non-viral vectors for the internalization of compounds such as peptides, oligonucleotides or proteins. Characterizing these carriers requires reliable methods to quantify their intracellular uptake. Flow cytometry on living cells is a method of choice but is not always applicable (e.g. big or polarized cells), so we decided to compare it to fluorescence spectroscopy on cell lysates. Surprisingly, for the internalization of a series of TAMRA-labeled conjugates formed of either cationic or amphipathic CPPs covalently coupled to a decamer peptide, we observed important differences in internalization levels between both methods.We partly explained these discrepancies by analyzing the effect of buffer conditions (pH, detergents) and peptide sequence/structure on TAMRA dye accessibility. Based on this analysis, we calculated a correction coefficient allowing a better coherence between both methods. However, an overestimated signal was still observable for both amphipathic peptides using the spectroscopic detection, which could be due to their localization at the cell membrane. Based on several in vitro experiments modeling events at the plasma membrane, we hypothesized that fluorescence of peptides entrapped in the membrane bilayer could be quenched by the tryptophan residues of close transmembrane proteins. During cell lysis, cell membranes are disintegrated liberating the entrapped peptides and restoring the fluorescence, explaining the divergences observed between flow cytometry and spectroscopy on lysates.Overall, our results highlighted major biases in the fluorescently-based quantification of internalized fluorescently-labeled CPP conjugates, which should be considered for accurate uptake quantification.

The consequences of adopting therapeutic luminophore azapodophyllotoxin into BSA: a molecular regulator to control emissive population of two tryptophan residues in carrier protein

Bovine serum albumin (BSA) is a widely recognized plasma protein for its ubiquitous function as one of the paramount transporter of different drugs and enzymes inside biological systems. HPFQ, a member of azapodophyllotoxin family, has been observed to be highly bioactive against a majority of cancer cell lines; while subsequently showing impressive fluorescent properties throughout the polarity scale. However, further pursuit into compliance of this bioactive fluorophore with carrier protein remains imperative for excavating its suitable transporter inside human body. The present biophysical spectroscopic study attempts to exhibit the adaptability of BSA towards a potential therapeutic fluorophore (HPFQ) by combining in vitro optical spectroscopy and in silico molecular docking. The competitive site-binding studies demonstrated that BSA nurtures neutral anti-cancer fluorophore HPFQ into Sudlow site I, where it experiences varying interactions with surrounding hydrophobic amino acid residues viz. Phe 205, Trp 213, Ala 209, Leu 330, Ala 349, Leu 480 etc. HPFQ gets accommodated at the vicinity of Trp-213 in BSA and initiates operation of FRET between them. Adaptation of HPFQ encourages an allosteric modulation, leading to a minor deformation in secondary protein structure, which probably allows the invading water molecules to increase the micropolarity of the adjacent environment around Trp-213. HPFQ assumes to administer conformational alteration in BSA and regulate emissive population of two tryptophan residues Trp-134 and Trp-213. The amalgamated spectroscopic investigation described herein may encourage design of azapodophyllotoxin based potential therapeutic agents for effective in vivo bio-circulation using BSA-based drug distribution systems.Communicated by Ramaswamy H. Sarma

Microenvironment of tryptophan residues in proteins of four structural classes: applications for fluorescence and circular dichroism spectroscopy

In this study we examined microenvironment of Trp residues in "dry" sets of nonhomologous proteins that belong to four structural classes, as well as in a "wet" set. In silico experiments showed that residues of Trp demonstrate higher surface accessibility in proteins of "alpha/beta" class where they are rarely included in beta strands. However, this feature has not caused "red" shift in fluorescence spectra in "alpha/beta" proteins in vitro, since there are several factors that should be combined together to cause it: high surface accessibility and high hydrophilicity of the microenvironment, the presence of destabilizing contacts with Asp, Asn, Leu, and multiple Tyr residues, as well as the lack of stabilizing interactions with Arg, Thr, and Pro. The occurrence of Trp residues has the highest value in beta-structural proteins, while they are not involved in aromatic-aromatic interactions with each other as frequently, as they do in proteins of "alpha + beta" class in which Trp residues are overrepresented near each other in the primary sequence. That is why the deformation of circular dichroism spectra because of Trp-Trp interactions is expected to be more frequent in proteins of "alpha + beta" class. In all four classes of proteins Trp residues are involved in long-range interactions with some hydrophobic (Leu, Val, Ile) and aromatic residues (Trp, Phe, and Tyr) more frequently than it is expected. They are involved in long-range interactions with some hydrophilic residues (Asp, Glu, Ser, and Lys) rarely than it is expected. Short-range interactions between Arg and Trp are overrepresented just in alpha-helical proteins.

Site-selective modification of tryptophan and protein tryptophan residues through PdNP bionanohybrid-catalysed C-H activation in aqueous media.

Herein we report for the first time the site-selective C-H bond arylation of tryptophan and tryptophan residues in proteins in aqueous media at room temperature by using a PdNP bionanohybrid as a heterogeneous catalyst. The reaction proceeds using a stable aryldiazonium salt without a base.

Biomolecular interactions of amphotericin B nanomicelles with serum albumins: A combined biophysical and molecular docking approach

In this work, we investigated the interaction of amphotericin B (AmB) nanomicelles on the binding affinity and conformational change of human serum albumin (HSA) in comparison with bovine serum albumin (BSA) under physiological conditions by conducting several spectroscopic techniques further confirmed through molecular docking approaches. The experimental results showed that AmB nanomicelles could bind to both HSA and BSA to form protein/drug complexes with one binding site, and the binding process was spontaneous under physiological conditions. Fluorescence studies revealed that the quenching mechanism of these complexes was static quenching rather than dynamic quenching and exhibited strong binding between serum albumin and AmB nanomicelles. The results from UV–Visible spectra, FT-IR spectra, and CD spectra revealed that the AmB formulations affected the structure of both HSA and BSA proteins by changing the microenvironment around the tryptophan residues of protein and caused a secondary structure change of the protein with the loss of helical stability. The molecular docking experiments also supported the above results and effectively proved the binding and changes in the conformation of serum albumins by AmB micelles. This finding provides information of in vitro drug-plasma protein interactions for further study on the AmB binding mechanism and the pharmacodynamics and pharmacokinetics.

Blue Death of Nematodes

This paper shows that the aging and death of nematodes, accompanied by the ignition of a blue glow under fluorescent microscopy, are not directly linked to any lipofuscin (aging pigment), nor with the anthranilic acid (a product of degradation of tryptophan residues of proteins). The main contribution in the blue flash of the dying nematodes belongs to parasitic light, scattered on the cuticle and bodies of the worm. The main contribution in the blue region at spectrofluorometry of homogenates, obtained from nematodes, really gives anthranilic acid. However, the content of anthranilic acid, measured by spectrofluorimetry, in adult nematodes is lower than that in the young ones. Artificial aging of nematodes by moderate heating revealed no accumulation of anthranilate and no loss of tryptophan, from which it must be formed. Thus, it is hardly lipofuscin or anthranilic acid. The cause of aging and death of nematodes is the formation of strong cross-links between proteins. This is supported by data on tryptophan fluorescence and light scattering of homogenates: the old worms show a large number of denaturated proteins and large protein particles with a strong cross-links, which are not destroyed be detergent.

Enrichment and characterization of a bacterial mixture capable of utilizing C-mannosyl tryptophan as a carbon source.

C-Mannosylation, a protein-modification found in various eukaryotes, involves the attachment of a single mannose molecule to selected tryptophan residues of proteins. Since C-mannosyl tryptophan (CMW) was detected in human urine, it is generally thought that CMW is not catabolized inside our body and instead is excreted via the urine. This paper reports enrichment of a bacterial consortium from soil that degrades CMW. The bacteria grew in minimal medium supplemented with CMW as the carbon source. Interestingly, even after successive clonal picks of individual colonies, several species were still present in each colony as revealed by 16S rRNA gene sequence analysis, indicating that a single species may not be responsible for this activity. A next generation sequencing (NGS) analysis was therefore carried out in order to determine which bacteria were responsible for the catabolism of CMW. It was found that a species of Sphingomonadaceae family, but not others, increased with simultaneous decrease of CMW in the media, suggesting that this species is most likely the one that is actively involved in the degradation of CMW.

A rapid and specific colorimetric method for free tryptophan quantification

Tryptophan is one of the eight essential amino acids and plays an important role in many biological processes. For its interaction with human health, environment and relevant commercial interest in biotechnology-based production, rapid and specific quantification method for this molecule accessible to common laboratories is badly needed. We herein reported a simple colorimetric method for free tryptophan quantification with 96-well-plate-level throughput. Our protocol firstly converted tryptophan to indole enzymatically by purified tryptophanases and then used reactivity of indole with hydroxylamine to form pink product with absorption peak at 530nm, enabling the quantification of tryptophan with simple spectrometry in just two hours. We presented that this method exhibited a linear detection range from 100μM to 600μM (R2 = 0.9969) with no detection towards other naturally occurring tryptophan analogs or tryptophan residues in proteins. It was very robust in complicated biological samples, as demonstrated by quantifying the titer of 36 mutated tryptophan-producing strains with Pearson correlation coefficient of 0.93 in contrast to that measured by high performance liquid chromatography (HPLC). Our method should be potent for routine free tryptophan quantification in a high-throughput manner, facilitating studies in medicine, microbiology, food chemistry, metabolic engineering, etc.

Sensing Tryptophan Microenvironment of Amyloid Protein Utilizing Wavelength-Selective Fluorescence Approach.

Structural transition among various forms of proteins involves subtle interplay between structure and dynamics and is crucial in human diseases. Red edge excitation shift (REES) represents a suitable approach to explore the environmental organization and dynamics surrounding tryptophan residues in proteins. Although REES from tryptophan residues has been reported for native, molten globule and denatured states of proteins, such data on the amyloid form of proteins is lacking. κ-casein is one of the most important constituents of casein micelles in milk and has a tendency to form amyloid fibril. We report here REES of the sole tryptophan residue for native, acid-denatured and urea-denatured forms of κ-casein. More importantly, we show that the amyloid form of κ-casein displays REES of 4nm. We analyze these results in terms of tryptophan microenvironment in various forms of κ-casein, particularly the amyloid form. We conclude that REES is a sensitive tool to monitor structural plasticity in proteins.