Tryptophan Residues In Proteins Research Articles

The Influence of Urea on the Structure of Proteins in Reversed Micelles

Static fluorescence measurements from the protein Tryptophan (Trp) residues and circular dichroism (CD) spectroscopy were used to investigate changes on the tertiary and secondary structures of the protein bovine serum albumin (BSA) and the dimeric enzyme hexokinase (HK) type PII from yeast, entrapped in reversed micelles (RMs). The latter were obtained from the amphiphilic AOT (sodium bis-2-ethylhexyl sulfosuccinate) in n-hexane, at several water to surfactant ratios, W. BSA and HK were found to be anchored at the RM interface in close contact with the surfactant layer, regardless of the size of the waterpool. For BSA, such interaction promotes partial protein unfolding, according to CD data that showed a decrease in the content of helical structure from 66% in a buffer solution to 48% in the micellar moiety. When urea was present in the micelle, further loss in helical structure occurred, thus indicating that the combined effect (micellar environment and urea) altered the BSA conformation to a greater extent than did RM or urea alone. Interestingly, Trps probed the same environment in the micelle, regardless of the presence of urea, but the fluorescence was quenched to a higher extent with urea. Thus, the fluorofore emission must have been affected either by the direct interaction of urea or by indirect exchange of water structure caused by urea interacting with water and the micellar interface. Both mechanisms might be of relevance in the solvation properties nearby the Trps. For HK, an association between the enzyme and the micelle interface was indicated in the CD spectra, which exhibited a randomized structure upon interaction, whatever the RM droplet size. The urea addition to the micelle water pool did not cause further impact on the HK conformation. In addition, the influence of urea at the RM interface was not sensed by the exposed tryptophans of the enzyme, unlike the results for BSA.

Use of fluorescence spectroscopy to differentiate yeast and bacterial cells

This study focuses on the characterization of bacterial and yeast species through their autofluorescence spectra. Lactic acid bacteria (Lactobacillus sp.), and yeast (Saccharomyces sp.) were cultured under controlled conditions and studied for variations in their autofluorescence, particularly in the area representative of tryptophan residues of proteins. The emission and excitation spectra clearly reveal that bacterial and yeast species can be differentiated by their intrinsic fluorescence with UV excitation. The possibility of differentiation between different strains of Saccharomyces yeast was also studied, with clear differences observed for selected strains. The study shows that fluorescence can be successfully used to differentiate between yeast and bacteria and between different yeast species, through the identification of spectroscopic fingerprints, without the need for fluorescent staining.

Label-Free Detection of Single Protein Molecules Using Deep UV Fluorescence Lifetime Microscopy

We present the detection of single beta-galactosidase molecules from Escherichia coli (Ecbeta Gal) using deep UV laser-based fluorescence lifetime microscopy. The native fluorescence from intrinsic tryptophan emission has been observed after one-photon excitation at 266 nm. Applying the time-resolved single-photon counting method, we investigated the fluorescence lifetime distribution and the bursts of autofluorescence photons from tryptophan residues in Ecbeta Gal protein as well as fluorescence correlation spectroscopy of Ecbeta Gal. The results demonstrate that deep UV laser-based fluorescence lifetime microscopy is useful for identification of biological macromolecules at the single-molecule level using intrinsic fluorescence.

Development of a rapid method based on front-face fluorescence spectroscopy for the monitoring of egg freshness: 2—evolution of egg yolk

This preliminary study is devoted to the application of front-face fluorescence spectroscopy to the study of egg yolks during storage. A total of 79 eggs stored for 1, 2, 3, 4, 5, 9, 10, 12, 16, 18, 23, 25 and 29 days at room temperature were analysed. The fluorescence emission spectra of tryptophan residues (excitation: 290 nm; emission: 305–430 nm) of proteins and the excitation spectra of vitamin A (emission: 410 nm; excitation: 270–350 nm) were recorded directly on egg yolk samples. Factorial discriminant analysis (FDA) was used to classify the eggs according to their date after they were laid. Using tryptophan fluorescence spectra, correct classification was observed for 57.1 and 51.9% for the calibration and the validation sets, respectively. Better classification (94.9 and 91.4% of the calibration and validation samples, respectively) was obtained from the vitamin A fluorescence spectra. The first five principal components (PCs) of the principal component analysis (PCA) extracted from each data set (tryptophan and vitamin A fluorescence spectra) were pooled (concatenated) into a single-matrix and analysed by FDA. Correct classifications were obtained for 97.5% of the calibration and 96.3.1% of the validation spectra. The discrimination of the investigated egg yolks according to their storage time was excellent. It was concluded that the concatenation of different fluorescence spectra might be considered as a promising indicator of shell egg freshness when they are used in egg products.

Development of a rapid method based on front face fluorescence spectroscopy for the monitoring of egg freshness: 1—evolution of thick and thin egg albumens

C � Springer-Verlag 2005 Abstract In this preliminary study, the intrinsic fluores- cence of thick and thin egg albumens was evaluated as a possible rapid method for the monitoring of egg freshness. The fluorescence emission spectra of tryptophan residues (excitation: 290 nm; emission: 305-430 nm) of proteins and fluorescent Maillard reaction products (excitation: 360; emission: 380-580 nm) were recorded directly on thick and thin albumen samples within 2-3, 4, 5, 9, 10, 12, 16, 18, 23, 25 and 29 days of storage. Principal component analy- sis (PCA) and factorial discriminant analysis (FDA) were applied to the spectra data sets. Considering tryptophan fluorescence spectra recorded on thick egg albumen, cor- rect classification was observed for 62.8 and 54.3% for the calibration and the validation sets, respectively. Better clas- sification was obtained from thin egg albumen since 67.3 and 69.1% of samples were correctly classified. Consid- ering fluorescent Maillard reaction products, the similarity map determined by the principal components (PCs) 1 and 2 showed a discrimination of eggs as a function of their storage time on both thick and thin albumens. The percent- age of samples correctly classified into four groups by the FDA was 97.4 and 91.4% for the calibration and validation thick albumen samples, respectively. It was concluded that fluorescent Maillard reaction products could be considered as fingerprints that may allow the discrimination between fresh and aged eggs.

Modification of tryptophan and tryptophan residues in proteins by reactive nitrogen species

Formation of 3-nitrotyrosine by the reaction between reactive nitrogen species (RNS) and tyrosine residues in proteins has been analyzed extensively and it is used widely as a biomarker of pathophysiological and physiological conditions mediated by RNS. In contrast, few studies on the nitration of tryptophan have been reported. This review provides an overview of the studies on tryptophan modifications by RNS and points out the possible importance of its modification in pathophysiological and physiological conditions. Free tryptophan can be modified to several nitrated products (1-, 4-, 5-, 6-, and 7-), 1-N-nitroso product, and several oxidized products by reaction with various RNS, depending on the conditions used. Among them, 1-N-nitrosotryptophan and 6-nitrotryptophan (6-NO(2)Trp) have been found as the abundant products in the reaction with peroxynitrite, and 6-NO(2)Trp has been the most abundant product in the reaction with the peroxidase/hydrogen peroxide/nitrite systems. 6-NO(2)Trp has also been observed as the most abundant nitrated product of the reactions between peroxynitrite or myeloperoxidase/hydrogen peroxide/nitrite and tryptophan residues both in human Cu,Zn-superoxide dismutase and in bovine serum albumin, as well as the reaction of peroxynitrite with myoglobin and hemoglobin. Several oxidized products have also been identified in the modified Cu,Zn-SOD. However, no 1-N-nitrosotryptophan and 1-N-nitrotryptophan has been observed in the proteins reacted with peroxynitrite or the myeloperoxidase/H(2)O(2)/nitrite system. The modification of tryptophan residues in proteins may occur at a more limited number of sites in vivo than that of tyrosine residues, since tryptophan residues are more buried inside proteins and exist less frequently in proteins, generally. However, surface-exposed tryptophan residues tend to participate in the interaction with the other molecules, therefore the modification of those tryptophans may result in modulation of the specific interaction of proteins and enzymes with other molecules.

Intramolecular Quenching of Tryptophan Phosphorescence in Short Peptides and Proteins¶

ABSTRACTThe phosphorescence lifetime (τ) of tryptophan (Trp) residues in proteins in aqueous solutions at ambient temperature can vary several orders of magnitude depending on the flexibility of the local structure and the rate of intramolecular quenching reactions. For a more quantitative interpretation of τ in terms of the local protein structure, knowledge of all potential quenching moieties in proteins and of their reaction rates is required. The quenching effectiveness of each amino acid (X) side chain and of the peptide backbone was investigated by monitoring their intramolecular quenching rate (kobs) in tripeptides of the form acetyl‐Trp‐Gly‐X‐CONH2 (WGX), where Trp is joined to × by a flexible Gly link. The results indicate that among the various groups present in proteins only the side chains of Cys, His, Tyr and Phe are able to quench Trp phosphorescence at a detectable rate (kobs > 40 s−1), with the quenching effectiveness for rotationally unrestricted side chains ranking in the order Cys >> His+ > Tyr >> Phe ∼ His. For the aromatic side chains the corresponding contact rate at 20°C is estimated to be between 3‐4 × 109 s−1 for Cys (as determined by Lapidus et al.), 0.8‐8 × 106 s−1 for His+, 0.37‐3.7 × 106 s−1 for Tyr and 0.2‐2 × 105 s−1 for Phe and His. In the cases of His and Tyr, kobs drops sharply with increasing pH, with midpoint transitions about 1 pH unit above the pKa, indicating that quenching is almost exclusive to the protonated form. From the temperature dependence of the rate, obtained in 50/50 propylene glycol/water between −20°C and 20°C, the reaction is characterized by activation energies of about 5 kcal·M−1 for His+ and Tyr and 8 kcal ·M−1 for Phe. An analysis of the groups in contact with Trp residues in proteins that exhibit long phosphorescence lifetimes at ambient temperature leads to the conclusion that the contact rate of the peptide group and of the remaining side chains is lower than 0.1 s−1, showing that these moieties are practically inert with respect to the triplet‐state lifetime. It shows further that the immobilization of the aromatic side chains within the globular fold cuts their quenching effectiveness drastically to contact rates < 2 s−1, a phenomenon attributed to the low probability of forming a stacked exciplex with the indole ring. All evidence suggests that, except in the case of nearby Cys or Trp residues, whose interaction with the triplet state reaches beyond van der Waals contact, the emission of buried Trp residues is unlikely to be quenched by surrounding protein groups.

Intramolecular quenching of Trp phosphorescence in short peptides and proteins

The phosphorescence lifetime (tau) of tryptophan (Trp) residues in proteins in aqueous solutions at ambient temperature can vary several orders of magnitude depending on the flexibility of the local structure and the rate of intramolecular quenching reactions. For a more quantitative interpretation of tau in terms of the local protein structure, knowledge of all potential quenching moieties in proteins and of their reaction rates is required. The quenching effectiveness of each amino acid (X) side chain and of the peptide backbone was investigated by monitoring their intramolecular quenching rate (k(obs)) in tripeptides of the form acetyl-Trp-Gly-X-CONH2 (WGX), where Trp is joined to X by a flexible Gly link. The results indicate that among the various groups present in proteins only the side chains of Cys, His, Tyr and Phe are able to quench Trp phosphorescence at a detectable rate (k(obs) > 40 s(-1)), with the quenching effectiveness for rotationally unrestricted side chains ranking in the order Cys >> His+ > Tyr >> Phe approximately His. For the aromatic side chains the corresponding contact rate at 20 degrees C is estimated to be between 3-4 x 10(9) s(-1) for Cys (as determined by Lapidus et al.), 0.8-8 x 10(6) s(-1) for His+, 0.37-3.7 x 10(6) s(-1) for Tyr and 0.2-2 x 10(5) s(-1) for Phe and His. In the cases of His and Tyr, k(obs) drops sharply with increasing pH, with midpoint transitions about 1 pH unit above the pKa, indicating that quenching is almost exclusive to the protonated form. From the temperature dependence of the rate, obtained in 50/50 propylene glycol/water between -20 degrees C and 20 degrees C, the reaction is characterized by activation energies of about 5 kcal.M(-1) for His+ and Tyr and 8 kcal.M(-1) for Phe. An analysis of the groups in contact with Trp residues in proteins that exhibit long phosphorescence lifetimes at ambient temperature leads to the conclusion that the contact rate of the peptide group and of the remaining side chains is lower than 0.1 s(-1), showing that these moieties are practically inert with respect to the triplet-state lifetime. It shows further that the immobilization of the aromatic side chains within the globular fold cuts their quenching effectiveness drastically to contact rates < 2 s(-1), a phenomenon attributed to the low probability of forming a stacked exciplex with the indole ring. All evidence suggests that, except in the case of nearby Cys or Trp residues, whose interaction with the triplet state reaches beyond van der Waals contact, the emission of buried Trp residues is unlikely to be quenched by surrounding protein groups.

Intramolecular Quenching of Tryptophan Phosphorescence in Short Peptides and Proteins¶

The phosphorescence lifetime (tau) of tryptophan (Trp) residues in proteins in aqueous solutions at ambient temperature can vary several orders of magnitude depending on the flexibility of the local structure and the rate of intramolecular quenching reactions. For a more quantitative interpretation of tau in terms of the local protein structure, knowledge of all potential quenching moieties in proteins and of their reaction rates is required. The quenching effectiveness of each amino acid (X) side chain and of the peptide backbone was investigated by monitoring their intramolecular quenching rate (k(obs)) in tripeptides of the form acetyl-Trp-Gly-X-CONH2 (WGX), where Trp is joined to X by a flexible Gly link. The results indicate that among the various groups present in proteins only the side chains of Cys, His, Tyr and Phe are able to quench Trp phosphorescence at a detectable rate (k(obs) > 40 s(-1)), with the quenching effectiveness for rotationally unrestricted side chains ranking in the order Cys >> His+ > Tyr >> Phe approximately His. For the aromatic side chains the corresponding contact rate at 20 degrees C is estimated to be between 3-4 x 10(9) s(-1) for Cys (as determined by Lapidus et al.), 0.8-8 x 10(6) s(-1) for His+, 0.37-3.7 x 10(6) s(-1) for Tyr and 0.2-2 x 10(5) s(-1) for Phe and His. In the cases of His and Tyr, k(obs) drops sharply with increasing pH, with midpoint transitions about 1 pH unit above the pKa, indicating that quenching is almost exclusive to the protonated form. From the temperature dependence of the rate, obtained in 50/50 propylene glycol/water between -20 degrees C and 20 degrees C, the reaction is characterized by activation energies of about 5 kcal.M(-1) for His+ and Tyr and 8 kcal.M(-1) for Phe. An analysis of the groups in contact with Trp residues in proteins that exhibit long phosphorescence lifetimes at ambient temperature leads to the conclusion that the contact rate of the peptide group and of the remaining side chains is lower than 0.1 s(-1), showing that these moieties are practically inert with respect to the triplet-state lifetime. It shows further that the immobilization of the aromatic side chains within the globular fold cuts their quenching effectiveness drastically to contact rates < 2 s(-1), a phenomenon attributed to the low probability of forming a stacked exciplex with the indole ring. All evidence suggests that, except in the case of nearby Cys or Trp residues, whose interaction with the triplet state reaches beyond van der Waals contact, the emission of buried Trp residues is unlikely to be quenched by surrounding protein groups.

Raman signature of the non-hydrogen-bonded tryptophan side chain in proteins: experimental and ab initio spectra of 3-methylindole in the gas phase

3-Methylindole (3MI), which serves as a structural model for the tryptophan side chain in proteins, has been investigated using vapor phase Raman spectroscopy. The vapor phase spectrum of 3MI identifies the Raman signature of the indolyl moiety free of intermolecular interaction and extends previously reported solution Raman studies of 3MI and related tryptophan derivatives. The Raman spectrum of 3MI vapor is also complemented here with newly obtained vapor phase infrared data and ab initio calculations to refine and extend previous vibrational assignments. The present results provide an improved basis for assessing the dependence of the indolyl Raman signature on the local environment of the tryptophan side chain of proteins. The principal conclusions of this work are the following. (i) The vapor phase 3MI molecule exhibits Raman bands at 3506, 1585, 1409, 1349/1341 (Fermi doublet) and 881cm−1, which differ greatly from their counterparts in the Raman spectrum of 3MI liquid and thus serve as spectral markers of the indolyl ring environment. (ii) The Fermi doublet relative intensity ratio (I1/I2, where I1 and I2 are, respectively, the Raman intensities of the higher and lower wavenumber components of the doublet) is highly sensitive to the state of 3MI condensation, consistent with the previously reported sensitivity of I1/I2 to solvent polarity. The maximum value of the intensity ratio (I1/I2=3.0) is observed for 3MI vapor, while the minimum value (I1/I2=0.43) is observed for 3MI in CHCl3 solution. Implications of the present results for Raman analysis of hydrogen bonding states, hydrophilic interactions and hydrophobic interactions of tryptophan residues in proteins are considered.

Rapid Purification and Mass Spectrometric Characterization of Mitochondrial NADH Dehydrogenase (Complex I) from Rodent Brain and a Dopaminergic Neuronal Cell Line

Oxidative stress and mitochondrial dysfunction signify important biochemical events associated with the loss of dopaminergic neurons in Parkinson's disease (PD). Studies using in vitro and in vivo PD models or tissues from diseased patients have demonstrated a selective inhibition of mitochondrial NADH dehydrogenase (Complex I of the OXPHOS electron transport chain) that affects normal mitochondrial physiology leading to neuronal death. In an earlier study, we demonstrated that oxidative stress due to glutathione depletion in dopaminergic cells, a hallmark of PD, leads to Complex I inhibition via cysteine thiol oxidation (Jha et al. (2000) J. Biol. Chem. 275, 26096-26101). Complex I is a approximately 980-kDa multimeric enzyme spanning the inner mitochondrial membrane comprising at least 45 protein subunits. As a prerequisite to investigating modifications to Complex I using a rodent disease model for PD, we developed two independent rapid and mild isolation procedures based on sucrose gradient fractionation and immunoprecipitation to isolate Complex I from mouse brain and a cultured rat mesencephalic dopaminergic neuronal cell line. Both protocols are capable of purifying Complex I from small amounts of rodent tissue and cell cultures. Blue Native gel electrophoresis, one-dimensional and two-dimensional SDS-PAGE were employed to assess the purity and composition of isolated Complex I followed by extensive mass spectrometric characterization. Altogether, 41 of 45 rodent Complex I subunits achieved MS/MS sequence coverage. To our knowledge, this study provides the first detailed mass spectrometric analysis of neuronal Complex I proteins and provides a means to investigate the role of cysteine oxidation and other posttranslational modifications in pathologies associated with mitochondrial dysfunction.

A Recombinant Glutamine-Binding Protein from Escherichia coli: Effect of Ligand-Binding on Protein Conformational Dynamics

We have investigated the effect of the binding of glutamine on the conformational dynamics of the recombinant glutamine binding protein (GlnBP) from Escherichia coli by steady-state and time-resolved fluorescence techniques. The structural stability of the protein was also studied by far-UV circular dichroism spectroscopy in the range of temperature between 25 and 80 degrees C. The results showed that the interaction of the protein with the ligand resulted in a marked change of the structural and conformational dynamics features of the protein. In particular, the fluorescence and circular dichroism data showed that the presence of glutamine resulted in a dramatic increase of the protein thermal stability of about 10 degrees C. In addition, the fluorescence time-resolved data pointed out that both in the absence and in the presence of glutamine the protein structure was highly rigid with small amplitude of segmental motion up to 65 degrees C and a low accessibility of the protein tryptophan residues to acrylamide. The obtained results on the structural properties of the recombinant glutamine-binding protein in the absence and in the presence of glutamine can contribute to a better understanding of the transport-related functions of the protein and structurally similar periplasmic transport proteins, as well as to the design and development of new biotechnological applications of this class of proteins.

The Effect of Bovine Serum Albumin on the Binding Constant and Stoichiometry of Biocompatible Magnetic Fluids

In this work, we investigated the interaction between different molecular-coated magnetite nanoparticles and the serum albumin protein. The investigation was based on the fluorescence quenching of the tryptophan residue of the serum albumin protein after the binding with the molecular-coated magnetite nanoparticles to specific sites. Three different biocompatible magnetic fluid samples based on magnetite nanoparticles surface-coated with carboxymethyldextran, tartrate, and polyaspartic were used. Significant differences in the values of binding constant (K/sub b/) and stoichiometry (n) were found as the surface-coating species are changed. The results obtained from the molecular-coated magnetite nanoparticles having different coatings indicate the effect of the coating material in the biological association of magnetite nanoparticles to biological macromolecules.

Nitration and nitrosation of N-acetyl- l-tryptophan and tryptophan residues in proteins by various reactive nitrogen species

Proteins are targets of reactive nitrogen species such as peroxynitrite and nitrogen dioxide. Among the various amino acids in proteins, tryptophan residues are especially susceptible to attack by reactive nitrogen species. We carried out experiments on the reactions of peroxynitrite and other reactive nitrogen species with N-acetyl- l-tryptophan under various conditions. Four major products were identified as 1-nitroso- N-acetyl- l-tryptophan, 1-nitro- N-acetyl- l-tryptophan, 6-nitro- N-acetyl- l-tryptophan, and N-acetyl- N′-formyl- l-kynurenine on the basis of their mass and UV spectra. The reactions with SIN-1 (a peroxynitrite generator), Angeli's salt (a nitroxyl donor), and spermine NONOate (a nitric oxide donor) generated the nitroso derivative but not the nitro derivatives. A myeloperoxidase–H 2O 2–NO 2 − system generated the nitro derivatives but not the nitroso derivative. Under physiological conditions 6-nitro- N-acetyl- l-tryptophan was stable, whereas the 1-nitroso and 1-nitro derivatives decomposed with half-lives of 1.5 and 18 h, respectively. After treatment with various reactive nitrogen species, bovine serum albumin was enzymatically hydrolyzed and analyzed for 6-nitro- l-tryptophan and 3-nitro- l-tyrosine by HPLC with electrochemical detection. Levels of 6-nitro- l-tryptophan and 3-nitro- l-tyrosine were similar in the nitrated protein. 6-Nitro- l-tryptophan in proteins can be measured as an additional biomarker of protein nitration.

Photo-CIDNP NMR methods for studying protein folding

Chemically induced dynamic nuclear polarization (CIDNP) is a nuclear magnetic resonance phenomenon that can be used to probe the solvent-accessibility of tryptophan, tyrosine, and histidine residues in proteins by means of laser-induced photochemical reactions, resulting in significant enhancement of NMR signals. CIDNP offers good sensitivity as a surface probe of protein structure and is particularly powerful in time-resolved NMR measurements. Real-time, rapid-injection protein refolding experiments permit the observation of changes in the accessibility of specific residues during the folding process. CIDNP pulse-labeling gives information on the accessibility of residues in partially structured proteins (e.g., molten globule states) whose NMR spectra are broad and poorly resolved. Heteronuclear two-dimensional 15N– 1H CIDNP techniques allow identification of surface-accessible residues with improved resolution and sensitivity. These methods offer residue-specific structural and kinetic information on transient folding intermediates and other partially folded states of proteins that are not readily available from more routine NMR techniques.

In vitro study on the interaction of mechanism of tricyclic compounds with bovine serum albumin

The mechanism of interaction of five phenothiazine drugs with bovine serum albumin has been investigated using fluorescence spectroscopy, circular dichroism and equilibrium dialysis methods. It was found that the phenothiazine ring common to all drugs makes major contribution to interaction. However, the nature of alkylamino group at position 10 influences the protein binding significantly. Binding affinities could be related to parachor values of drugs. Stern–Volmer plots indicated the presence of static component in the quenching mechanism. Results also showed that both tryptophan residues of protein are accessible to drug molecules. The high magnitude of rate constant of quenching indicated that the process of energy transfer occurs by intermolecular interaction forces and thus drug-binding site is in close proximity to tryptophan residues of BSA. Binding studies in presence of hydrophobic probe, 8-anilino-1-naphthalein-sulphonic acid showed that there is hydrophobic interaction between drugs and the probe and they do not share common sites in BSA. Fluorescence intensity data in the presence of additives showed that hydrophobic interactions play a significant role. Small decrease in critical micellar concentration of anionic surfactant, sodium dodecyl sulfate in the presence of drugs showed that the ionic character of drugs also contribute to binding. Thermodynamic parameters obtained from data at different temperatures showed that the binding of phenothiazine drugs to BSA involve hydrophobic bonds predominantly. The CD spectrum of BSA in presence of drugs shows that binding of drugs leads to change in the helicity of the protein. The binding of these drugs to BSA based on dialysis experiment has been characterized by association constant ( K) and the number of binding sites ( n).

Resolving near-ultraviolet circular dichroism spectra of single trp mutants in tear lipocalin

Near-ultraviolet circular dichroism (near-UV CD) spectra of tryptophan residues in proteins are complicated because the line shapes are derived from the overlap of both the 1L a and the 1L b electronic bands that vary independently. Contributing to this complexity, tryptophan near-UV CD spectra differ in the relative amplitude of the 0-0 vibronic band compared to the rest of the 1L b spectrum, an inherent feature that may result in poor fitting. To resolve this problem, a computer program that incorporated the separation of the 0-0 transition of 1L b component from the rest of the 1L b was written in LabVIEW and its amplitude was allowed to vary independently. This method showed dramatically improved fitting of 1L a and 1L b components in the near-UV CD tryptophan spectra in tear lipocalin mutants featuring low intensity of the 0-0 1L b component. Side chain dynamic characteristics (mobility and accessibility to the solvent) identified from different spectroscopic techniques were related to differences in Trp near-UV CD spectra. This method is broadly applicable to different types of Trp near-UV CD spectra.

Oxidative Post-translational Modification of Tryptophan Residues in Cardiac Mitochondrial Proteins

We examined the distribution of N-formylkynurenine, a product of the dioxidation of tryptophan residues in proteins, throughout the human heart mitochondrial proteome. This oxidized amino acid is associated with a distinct subset of proteins, including an over-representation of complex I subunits as well as complex V subunits and enzymes involved in redox metabolism. No relationship was observed between the tryptophan modification and methionine oxidation, a known artifact of sample handling. As the mitochondria were isolated from normal human heart tissue and not subject to any artificially induced oxidative stress, we suggest that the susceptible tryptophan residues in this group of proteins are "hot spots" for oxidation in close proximity to a source of reactive oxygen species in respiring mitochondria.

Development of a rapid method based on front-face fluorescence spectroscopy for the monitoring of fish freshness

The intrinsic fluorescence of fish muscle was evaluated as a possible rapid and non-destructive method for the monitoring of fish freshness. The fluorescence emission spectra of aromatic-amino-acids+nucleic-acids (excitation: 250 nm, emission: 280–480 nm), tryptophan residues (excitation: 290 nm, emission: 305–400 nm) of proteins and NADH (excitation: 336 nm, emission: 360–600 nm) were recorded on cod, mackerel, salmon and whiting fillets at 1, 5, 8 and 13 days of storage. Principal component analysis and Mahalanobis distance method were applied to the spectral data sets. Considering the mackerel, the similarity map defined by the principal components 1 and 2 showed a discrimination of the aromatic-amino-acids+nucleic-acids spectra recorded after 1 day of storage versus the ones recorded after 5 and 8 days of storage according to the principal component 1. Similar trends were observed for all the fish species and all the fluorophores investigated. It appears that the intrinsic fluorescence spectra could be considered as fingerprints that may allow the discrimination between fresh and aged fish fillets.

Structural features of proteins responsible for resistance of tryptophan residues to nitrosylation.

It is known that potentially reactive groups of the protein molecule may be most efficiently nitros(yl)ated only when located within hydrophobic globules or built into the membrane. N1-nitrosotryptophan (NOW) is a stable product of nitrosation in vitro. However, the NOW fraction in proteins is small in ordinary proteins. It suggests the existence of unknown mechanisms preventing the accumulation of NOW. Here we show that these mechanisms are underlain by the protein structure. Analysis of protein structure databases to explore the atomic surroundings of tryptophan residues revealed preferential selection of certain surroundings. N(E) atoms of tryptophan residues, which are the targets for nitrosation, have usually polar and nucleophilic groups in their environment. Residues of Asp, Glu, Cys, His, and Met act as catalysts of denitrosation (internal denitrosilase). We found that short peptides with the same residues possessed denitrosilase activity even in solution. This selection might explain both the resistance of tryptophan residues in proteins to nitrosation and the mechanisms of chemical communication by means of reversible nitrosation of proteins.