Tryptophan Residues In Lysozyme Research Articles

Unveiling the Influence of Hydrated Deep Eutectic Solvents on the Dynamics of Water-Soluble Proteins.

Deep eutectic solvents (DESs) are mixtures of two or more pure compounds (e.g., Lewis or Brønsted acids and bases, anionic and/or cationic species) in a well-defined stoichiometric proportion, with a melting point lower to that of an ideal liquid mixture. These neoteric solvents are highly tunable through varying the structure or relative ratio of parent components and have been evaluated as solvents able to improve biomolecules' performance, specifically their stability and biocatalytic properties. Inspired by a recent crystallographic study, we have explored through molecular dynamics (MD) simulations the dynamic properties of two different proteins (hen egg-white lysozyme and the human VH antibody fragment HEL4) in a (20% w/w) hydrated solution of choline chloride-glycerol (1:2). We have developed proper force fields to account for DES, protein, and DES-protein interactions, which have been calibrated using pair distribution function measurements of pure DES solutions. MD results show that the presence of DES quenches the protein motion, increasing the rigidity of the overall protein structure. Specific interactions among DES components and protein residues, such as those between choline ions and two Tryptophan residues of lysozyme, may amplify the protein-DES interactions and lead to protein crystallization in the presence of hydrated DES. These findings open new horizons to improve or achieve control on protein properties by a proper choice of hydrated DESs used as solvents.

The study on interactions between levofloxacin and model proteins by using multi-spectroscopic and molecular docking methods

The interactions of levofloxacin (LEV) with lysozyme (LYZ), trypsin and bovine hemoglobin (BHb) were investigated, respectively, by using multi-spectral techniques and molecular docking in vitro. Fluorescence studies showed that LEV quenched LYZ/trypsin fluorescence in a combined quenching ways and BHb fluorescence in a static quenching with binding constants of .14, .51 and .20 × 105 L mol−1 at 298 K, respectively. The thermodynamic parameters demonstrated that hydrophobic forces, hydrogen bonds, and van der Waals forces played the major role in the binding process. The binding distances between LEV and the inner tryptophan residues of LYZ, trypsin, and BHb were calculated to be 4.04, 3.38, and 4.52 nm, respectively. Furthermore, the results of circular dichroism spectra (CD), UV–vis, and three-dimensional fluorescence spectra indicated that the secondary structures of LYZ, trypsin, and BHb were partially changed by LEV with the α-helix percentage of LYZ-LEV system increased while that of BHb-LEV system was decreased, the β-sheet percentage of trypsin-LEV system increased from 41.3 to 42.9%. UV–vis spectral results showed that the binding interactions could cause conformational and some micro-environmental changes of LYZ, trypsin, and BHb. The results of molecular docking revealed that in LYZ and trypsin systems, LEV bound to the active sites residues GLU 35 and ASP 52 of LYZ and trypsin at the active site SER 195, and in BHb system, LEV was located in the central cavity, which was consistent with the results of synchronous fluorescence experiment. Besides, LEV made the activity of LYZ decrease while the activity of trypsin increased.

Formation of 6-nitrotryptophan in purified proteins by reactive nitrogen species: A possible new biomarker

Abstract We analyzed products of tryptophan residues in two model proteins which were reacted with reactive nitrogen species. We modified human Cu, Zn-superoxide dismutase, which has a single tryptophan residue and no tyrosine residue, by using two reactive nitrogen species generating systems; peroxynitrite/CO 2 and myeroperoxidase/H 2 O 2 /NO 2 − systems. We identified 6-nitrotryptophan as a major nitration product along with other oxidized products as the reaction products of tryptophan residue by using LC-MS/MS and HPLC-photodiode array analyses of the tryptic peptides. We modified hen egg-white lysozyme as a model of a simple protein having both tryptophan and tyrosine residues by peroxynitrite/CO 2 system. The modified enzyme lost 89% of the enzymatic activity. Among six tryptophan residues in lysozyme, Trp62, Trp63, and Trp123 were nitrated to form 6-nitrotryptophan, along with the formation of 3-nitrotyrosine in all tyrosine residues. However, the efficiency of nitration was different for each residue. No oxidized product of tryptophan residue was observed in the modified lysozyme. In conclusion, we propose that 6-nitrotryptophan is a unique and major nitrated product of tryptophan residue in proteins reacted with reactive nitrogen species.

Resonance energy transfer study of lysozyme–lipid interactions

Resonance energy transfer (RET) between the tryptophan residues of lysozyme as donors and anthrylvinyl-labeled phosphatidylcholine (AV-PC) or phosphatidylglycerol (AV-PG) as acceptors has been examined to gain insight into molecular level details of the interactions of lysozyme with the lipid bilayers composed of PC with 10, 20, or 40 mol% PG. Energy transfer efficiency determined from the enhanced acceptor fluorescence was found to increase with content of the acidic lipid and surface coverage. The results of RET experiments performed with lipid vesicles containing 40 mol% PG were quantitatively analyzed in terms of the model of energy transfer in two-dimensional systems taking into account the distance dependence of orientation factor. Evidence for an interfacial location of the two predominant lysozyme fluorophores, Trp62 and Trp108, was obtained. The RET enhancement observed while employing AV-PG instead of AV-PC as an energy acceptor was interpreted as arising from the ability of lysozyme to bring about local demixing of the neutral and charged lipids in PC/PG model membranes.

Propagation of protein glycation damage involves modification of tryptophan residues via reactive oxygen species: inhibition by pyridoxamine

Nonenzymatic modification of proteins is one of the key pathogenic factors in diabetic complications. Uncovering the mechanisms of protein damage caused by glucose is fundamental to understanding this pathogenesis and in the development of new therapies. We investigated whether the mechanism involving reactive oxygen species can propagate protein damage in glycation reactions beyond the classical modifications of lysine and arginine residues. We have demonstrated that glucose can cause specific oxidative modification of tryptophan residues in lysozyme and inhibit lysozyme activity. Furthermore, modification of tryptophan residues was also induced by purified albumin–Amadori, a ribose-derived model glycation intermediate. The AGE inhibitor pyridoxamine (PM) prevented the tryptophan modification, whereas another AGE inhibitor and strong carbonyl scavenger, aminoguanidine, was ineffective. PM specifically inhibited generation of hydroxyl radical from albumin–Amadori and protected tryptophan from oxidation by hydroxyl radical species. We conclude that oxidative degradation of either glucose or the protein–Amadori intermediate causes oxidative modification of protein tryptophan residues via hydroxyl radical and can affect protein function under physiologically relevant conditions. This oxidative stress-induced structural and functional protein damage can be ameliorated by PM via sequestration of catalytic metal ions and scavenging of hydroxyl radical, a mechanism that may contribute to the reported therapeutic effects of PM in the complications of diabetes.

Study on the interaction of puerarin with lysozyme by spectroscopic methods

The interaction between lysozyme (LYSO) and puerarin has been studied at three temperatures (294, 302 and 310 K) through/using fluorescence spectroscopy and circular dichroism (CD). The LYSO fluorescence was quenched by the binding of puerarin to LYSO. The binding constants and the number of binding sites can be calculated from the data obtained from fluorescence quenching experiments. According to the van’t Hoff equation, the standard enthalpy change (Δ H°) and standard entropy change (Δ S°) for the reaction were calculated to be 17.47 kJ/mol and 163.5 J/mol K. It indicated that the hydrophobic interactions play a main role in the binding of puerarin to LYSO. In addition, the distance between puerarin (acceptor) and tryptophan residues of LYSO (donor) was estimated to be 1.47 nm on the basis of fluorescence energy transfer. The changes of LYSO secondary structure in the presence of puerarin were observed from CD spectroscopy.

Internal motion of lysozyme studied by time-resolved fluorescence depolarization of tryptophan residues.

The internal motion of lysozyme was described by the steady-state and time-resolved fluorescence anisotropy of its tryptophan residues. The fluorescence of mutant lysozymes W62Y- and W108Y-lysozyme, in which Trp62 or Trp108 of hen egg white lysozyme was replaced with a tyrosine residue, could be respectively assigned to Trp108 or Trp62 at the longer wavelength region of the total fluorescence spectrum. The segmental motion of Trp62 as shown by its fluorescence anisotropy decay was described with two components originating from the fluctuational rotation of an indole moiety about the Calpha-Cbeta bond and rotational wobble of the peptide segment adjacent to Trp62. Although Trp62 showed a high degree of motional freedom, its motion was significantly suppressed by the interaction of the mutant protein with a trimer of N-acetyl-D-glucosamine. By contrast, the segmental motion of Trp108 is hindered by the local cage structure at temperatures below 30 degreesC, but Relief from restricted motion occurred on the formation of ligand complex or by thermal agitation. Because of overlaps of the fluorescence spectrum, it is difficult to assign the segmental motion of Trp28 or Trp111, the other two tryptophan residues in lysozyme. However, a careful analysis of the fluorescence anisotropy decay of W62Y- and W108Y-lysozyme showed that the fluctuation of the hydrophobic matrix box was greater than that expected from lysozyme's crystal structure, although it was suppressed by the binding of the ligand to the active site of lysozyme.

The effects of tyrosine nitration on the structure and function of hen egg-white lysozyme.

We have investigated the effects of tyrosine nitration (to form the weak acid, 3-nitrotyrosine) at positions 23 or 20 plus 23, on the structure and function of hen egg-white lysozyme. Enzyme activity against Micrococcus luteus cell-wall fragments or soluble substrates exhibits two phenomena. (a) A decrease in Km and kcat for the hydrolysis of soluble oligo- and poly-saccharides, resulting in only minor changes in the catalytic efficiency (kcat/Km) upon nitration. (b) The hydrolysis of M. luteus cell-wall fragments appeared to be dominated by electrostatic interactions with the protein, giving a decrease in enzyme activity as the 3-nitrotyrosyl group became ionized. Removal of the cell-wall anionic polymer, teichuronic acid, from M. luteus abolished this effect. The 3-nitrotyrosine group was also found to act as a fluorescence quencher of exposed tryptophan residues in lysozyme.

Quantitative fluorescence analysis of the adsorption of lysozyme to phospholipid vesicles

Experiments directed to measure the interaction of lysozyme with liposomes consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) have been conducted by monitoring both protein and lipid fluorescence and fluorescence anisotropy of the protein. The binding of lysozyme to the unilamellar vesicles was quantified using a novel method of analysis in which the fractional contribution at moderate binding conditions is determined from either total fluorescence decay or anisotropy decay curves of tryptophan at limiting binding conditions. In the energy transfer experiments PC and PS lipids labelled with two pyrene acyl chains served as energy acceptors of the excited tryptophan residues in lysozyme. The binding was strongly dependent on the molar fraction of negatively charged PS in neutral PC membranes and on the ionic strength. Changes in the tryptophan fluorescence decay characteristics were found to be connected with long correlation times, indicating conformational rearrangements induced by binding of the protein to these lipid membranes. The dynamics of membrane bound protein appeared to be dependent on the physical state of the membrane. Independent of protein fluorescence studies, formation of a protein-membrane complex can also be observed from the lipid properties of the system. The interaction of lysozyme with di-pyrenyl-labelled phosphatidylserine in anionic PS/PC membranes resulted in a substantial decrease of the intramolecular excimer formation, while the excimer formation of dipyrenyl-labelled phosphatidylcholine in neutral PC membranes barely changed in the presence of lysozyme.

Quasielastic light scattering, electrophoresis, and fluorescence studies of lysozyme‐poly(2‐acrylamido‐methylpropylsulfate) complexes

AbstractComplexation between lysozyme and sodium poly(2‐acrylamido‐2‐methylpropanesulfonate) (PAMPS) was studied by quasielastic light scattering, electrophoretic light scattering, fluorescence, and turbidimetry in electrolyte solution. These techniques show that complexation occurs at pH 9.6 in an ionic strength buffer of 0.25M NaCl + 25 mM Na2B4O7. At constant lysozyme concentration (Cpro). The structure of the complex depends on the polymer concentration. At low polymer concentration (relative to Cpro), an intrapolymer complex is formed. This intrapolymer complex aggregates to an interpolymer species upon increase in polymer concentration. Complex formation was also studied by fluorescence using pyrene‐labeled PAMPS (Py‐PAMPS). Energy transfer from singlet‐excited tryptophan residues in lysozyme to the pyrene label occurs when the complex is formed. Fluorescence and turbidity data indicate that lysozyme interacts with Py‐PAMPS preferentially at pyrene sites, which leads to static quenching of tryptophan fluorescence via energy transfer to the pyrene label. © 1995 John Wiley & Sons, Inc.

Hydration and protein substates: Fluorescence of proteins in reverse micelles

Abstract The fluorescence properties of indole derivatives, lysozyme and azurin were investigated in reverse micelles of detergent sodium bis[2-ethylhexl]sulfosuccinate (Aerosol OT)* in n-hexane. L-tryptophan, l-methyl-tryptophan and n-acetyl-l-tryptophanamide exhibited complex fluorescence decays in reverse micelles. Fluorescence decays were best described using Gaussian bimodal distributions of lifetimes. Increasing hydration levels in the micelles resulted in a decrease in decay heterogeneity, as indicated by a large decrease in lifetime distribution widths. Steady-state polarization and fluorescence emission measurements indicated both an increase in average polarity of the environment around the indole derivatives and an increase in the mobility of the probes with increasing hydration levels. The fluorescence decays of lysozyme and azurin in reverse micelles were also found to be very complex and were described with Gaussian lifetime distributions. Increasing water content in the micelles caused marked decreases in both center and width of the lifetime distributions for these two proteins. Steady-state polarization measurements as a function of the extent of hydration revealed an increase in the average rotational rates of the tryptophan residues in lysozyme upon increasing water content. Thus, static polarization and lifetime measurements indicate that the amount of water present in the micelle may influence the amount of structural flexibility of the polypeptide chains and the rates of interconversion between conformational substates.

Proton nuclear magnetic resonance assignments and surface accessibility of Tryptophan residues in lysozyme using photochemically induced dynamic nuclear polarization spectroscopy.

Tryptophan resonances in the 360-MHz 1H photochemically induced dynamic nuclear polarization spectrum of hen egg white lysozyme are investigated in detail. All resonances of one tryptophan and six of another are identified and assigned to their respective protons. The methods employed, all involving nuclear spin polarization, include the study of cross-relaxation effects and the use of selective radio-frequency irradiation, Gd3+ as a paramagnetic probe, and riboflavin as the chemically induced dynamic nuclear polarization generating dye. From a comparison of the experimental results with the known X-ray structure of lysozyme, second-stage assignments of the two tryptophan residues (Trp-62 and Trp-123) are proposed. A number of other resonances are characterized, among them Trp-63 C(2)H and four indirectly polarized methyl groups.

Rates of structural fluctuations of lysozyme in the range of thermal unfolding transition.

AbstractThe hydrogen–deuterium exchange reaction for the tryptophan residues in lysozyme have been followed in 4.5M LiBr at pH 7.2 in the temperature range of the unfolding transition by measuring the transmittance change at 293 nm. The exchange reaction proceeded in three phases at low temperature for native protein. The first and the second phases were ascribed to the H‐D exchange reactions of three relatively exposed tryptophan residues on the molecular surface. The third phase corresponded to the H‐D exchange reaction of the three tryptophan residues buried in the interior of the molecule. The H‐D exchange reaction proceeded in two phases near the melting temperature and in a single phase at high temperature, where almost all molecules are unfolded. The H‐D exchange of three tryptophan residues buried in folded molecules was caused by fluctuation between the folded and unfolded structure of the protein molecule. The rates of such a fluctuation were determined from the rates of the exchange reaction at various temperatures. These rates agreed very well with those determined from the temperature‐jump method. This means that a protein molecule in solution fluctuates between the N‐ and D‐states at every temperature within the transition region, where the N‐form is the tightly folded native structure and the D‐form the randomly coiled chain. From measurements of thermal unfolding of ester‐108‐lysozyme and the binding constant of (NAG)3 to ester‐108‐lysozyme, it was found that almost all cross‐linked molecules are in the folded state near 50°C and pH 7.2 in 4.5M LiBr, where intact molecules are unfolded. We also studied the H‐D exchange reaction of ester‐108‐lysozyme. In the temperature region of 43–50°C, about 70% of the exchangeable tryptophan residues of ester‐108‐lysozyme were exchanged within 1 s immediately after the mixing of D2O, in spite of the fact that almost all molecules are in the folded state. This was considered the premelting of the surface of a corss‐linked molecule.

Triplet state of tryptophan in proteins. 2. Differentiation between tryptophan residues 62 and 108 in lysozyme

We have used optically detected magnetic resonance (ODMR) to characterize the degree of solvent availability of the tryptophan residues in lysozyme that are likely to be responsible for the observed phosphorescence. From the phosphorescence spectra, ODMR zero-field splittings (zfs), and ODMR line widths, we concur with the X-ray structure [Blake, C. C., Mair, G. A., North, A. C. T., Phillips, D. C., & Sarma, V. R. (1967) Proc. R. Soc. London, ser. B 167, 365-377] that Trp-62 behaves as an exposed residue and Trp-108 is buried. In addition, we present evidence that ODMR can be used in conjunction with conventional phosphorescence to evaluate the degree of order in the microenvironments of tryptophan in a protein containing several tryptophans. By the specific modification of residues Trp-62 and Trp-108, we have identified those portions of the ODMR lines in the native enzyme that are due to those specific residues. Barring major enzyme conformational changes in the vicinity of unmodified tryptophan residues when Trp-62 or Trp-108 are selectively modified, we find that Trp-108 dominates both the phosphorescence and the ODMR signals in native lysozyme. The results are discussed in view of previous fluorescence findings.

The inhibition of hen egg-white lysozyme by imidazole and indole derivatives

The inhibition of hen egg-white lysozyme by various imidazole and indole derivatives has been examined by kinetic and crystallographic methods. The results are consistent with the formation of charge-transfer complexes between the imidazole derivatives and tryptophan residues in lysozyme. In contrast, indole-3-propionic acid enters a hydrophobic region of the protein, leading apparently to distortion of the active site, 10 to 15 Å away, with concomitant loss of activity. The comparison of the kinetic and crystallographic data suggests that the direct interpretation of inhibition studies is open to question when the inhibitor is not a close analogue of the in vivo substrate.

Enzymic and immunochemical properties of lysozyme — II.: Conformation, immunochemistry and enzymic activity of a derivative modified at tryptophan

The six tryptophan residues in lysozyme were modified by reaction for 3 min with 2-nitrophenyl sulfenyl chloride in 98 per cent formic acid. The modified enzyme showed a sedimentation coefficient (S 0 20, w ) of 4.05 S compared to 1·91 S for native enzyme and 1·95 S for formic acid-treated lysozyme. Significant conformational changes accompanied the modification as evaluated by the availability of the disulfide linkages to reduction and the susceptibility of the modified protein to proteolysis. Loss of enzymic activity as well as a marked decrease in the ability of modified lysozyme to react with antilysozyme antibodies were observed. There was evidence that modified lysozyme showed decreased antigenicity in rabbits.

States of amino acid residues in proteins: V. Different reactivities with H 2O 2 of tryptophan residues in lysozyme, proteinases and zymogens

Hydrogen and organic peroxides were examined in a search for a reagent to differentiate between various states of tryptophan residues in proteins. Hydrogen peroxide in a solution of 0.5 M bicarbonate buffer (pH 8.1–9.4) containing 10% dioxane was found to possess a moderate oxidizing power to oxidize free and bound tryptophan residues at different concentrations of H 2O 2, and the strength of the tryptophan absorption band of the protein was lowered stepwise with increasing concentration. By the use of this reagent, tryptophan residues in several proteins were classified into various types with different oxidizabilities, and the moles, n, of each type per mole of proteine were determined; n=5 and 1 for lysozyme (EC 3.2.1.17), n=5, 1 and 1 for chymotrypsinogen and α-chymotrypsin (EC 3.4.4.5), and n=2, 1 and 1 for trypsinogen and trypsin (EC 3.4.4.4.) which are arranged in the order of decreasing oxidizability. On the activation of chymotrypsinogen, the oxidizability of the most strongly bound residue decreased greatly whereas, on the activation of trypsinogen, the oxidizability of the secondly oxidized residue increased slightly. The structural rearrangement on the activation was discussed in terms of these changes in state of tryptophan as well as those of other amino acids determined previously. The enzymic activity of lysozyme an proteinases was also measured as a function of H 2O 2 concentration, and the results were correlated with the degree of oxidation of tryptophan residues. All of the tryptophan residues in denatured proteins were oxidized at a low concentration of H 2O 2, so that the reagent is applicable for the determination of the molar tryptophan content in protein.