Tryptophyl Residues In Proteins Research Articles

Ring-Toss: Capping highly exposed tyrosyl or tryptophyl residues in proteins with β-cyclodextrin

We have used UV difference spectroscopy and fluorescence spectroscopy to study the perturbation by beta-cyclodextrin of tyrosyl or tryptophyl residues located at each of the 10 variable consensus contact positions in the third domain of turkey ovomucoid. The goal was to monitor the accessibility of the side chain rings of these residues when located at these positions. The results indicated that the tyrosyl or tryptophyl rings are most highly exposed when located in the P1 position followed by the P4 position. It was possible to determine the association constants for beta-cyclodextrin binding at these positions. When located at the P2, P5, P6 and P3' positions, the rings of the tyrosyl or tryptophyl residues were exposed but less so than at the P1 or P4 positions. By contrast, when located at the P1', P2', P14' and P18' positions, the tyrosyl or tryptophyl residues were insufficiently exposed to be perturbed by beta-cyclodextrin, although they reacted positively to dimethyl sulfoxide solvent perturbation. These findings indicate that beta-cyclodextrin perturbation provides a convenient way to detect highly exposed tyrosyls or tryptophyls in proteins. Furthermore, we evaluated the ability of beta-cyclodextrin to inhibit the interaction of turkey ovomucoid third domain variants with different P1 residues. The results showed that the presence of beta-cyclodextrin had little effect on the association constant when the P1 residue was a glycyl residue, but greatly decreased the association constant when the P1 residue was a tyrosyl or tryptophyl residue. Thus, beta-cyclodextrin may be used to selectively modulate the interaction between proteinase inhibitors and their cognate enzymes.

A new component in protein fluorescence.

Tryptophyl residues in proteins absorb at longer wavelengths than tyrosyl residues, thus the tryptophyl fluorescence can be selectively excited. In addition, tryptophyl residues have a fluorescence maximum at much longer wavelengths than tyrosyl residues and are the predominant source of fluorescence in the long-wavelength region. The contribution of tyrosyl fluorescence to protein fluorescence can be determined by exploiting these spectral properties. The fluorescence of the tyrosyl residues in native human serum albumin is different from the fluorescence of N-acetyl-L-tyrosinamide; the spectral maximum is at a longer wavelength, and the spectral width is greater. This difference is caused by a second component with a maximum at 345 nm. The excitation spectrum of the 345-nm component is similar to that of the normal 304-nm tyrosyl component. The 345-nm component is largely absent from denatured serum albumin. An excited singlet state protolysis from the buried tyrosyl residues explains the appearance of the 345-nm component. A possible acceptor base is an amino group of buried lysyl residue.

REACTION OF NITRITE WITH TRYPTOPHYL RESIDUES OF PROTEIN

ABSTRACTThe reaction of sodium nitrite with tryptophyl residues of protein was investigated. A reaction was not observed when native myosin was treated with nitrite at pH 6.0. When denatured myosin was reacted with nitrite at pH 6.0, some response was seen, and at pH 3.0, evidence (although weak) was obtained that tryptophyl residues were nitrosated. Spectrophotometric evidence for nitrosation of tryptophyl residues was obtained when lysozyme was treated with nitrite; the reaction ZBS promoted by low pH, by heat and by increasing concentration of nitrite. We concluded that while the nitrosation of tryptophyl residues in proteins can take place, the conditions in cured meat are such that if the reaction did occur, the amount of product formed would be extremely small.

Site heterogeneity of tryptophyl residues in proteins determined by fluorescence quenching [proceedings

Site heterogeneity of tryptophyl residues in proteins determined by fluorescence quenching [proceedings

Kinetics of hydrogen-deuterium exchange of tryptophan and tryptophan peptides in deutero-trifluoroacetic acid using proton magenetic resonance spectroscopy.

The methods which have been used for the observation and assignment of resonances in the NMR spectra of proteins are reviewed. One such method, the selective deuteration of the aromatic protons of tryptophyl residues, is studied by NMR spectroscopy in model compounds in this paper, and in proteins in the following paper. On the basis of a reassignment of the PMR spectrum of the aromatic protons of L-tryptophan, the relative rates of H-D exchange in deutero-trifluoracetic acid (d-TFA) are H-2 greater than H-5 greater than H-6 greater than H-4 approximately H-7. The energies of activation for the first order exchange of both the H-2 and H-5 protons is 12 k.cal.mol-1. The rate constant for exchange of the H-2 protons of tryptophyl residues in peptides is much greater than in the amino acid itself and 5-10 times that for exchange of the H-5 protons. This suggests that the method can be used to label tryptophyl residues in proteins rapidly and specifically.

QUENCHING OF TRYPTOPHYL FLUORESCENCE IN PROTEINS BY N‘‐METHYLNICOTINAMIDE CHLORIDE*

Abstract— N‘‐methylnicotinamide chloride has been investigated as a quencher of the intrinsic fluorescence of tryptophyl residues in proteins. Quenching of the tryptophyl fluorescence (excitation at 305 nm) of model compounds and a select group of proteins provide evidence for a mechanism that follows the classical Stern‐Volmer relation. The apparent number of exposed tryptophyl residues was obtained in 6M guanidine hydrochloride and aqueous solution at constant pH. Evidence that quenching occurs predominantly by a collisional mechanism was obtained by comparing the equilibrium constant of the non‐fluorescent static state with the much larger quenching constant of the excited state.

The application of enzymic hydrolysis and tritium labelling to a study of the modification of tryptophyl residues in proteins.

The tryptophyl residues of apomyoglobin, S-carboxymethyl-Iysozyme, and wool have been radioactively labelled in order to follow their reactions more easily. Whereas enzymic hydrolysis of these proteins gives good yields of tryptophan, it was found that enzymic hydrolysis of apomyoglobin, after either ozonolysis or treatment with o-nitrophenylsulphenyl chloride, released only part of the resultant modified tryptophyl residues.

Tritiation of tryptophyl residues in proteins.

Tritiation of tryptophyl residues in proteins.

The reaction of dimethyl (2-hydroxy-5-nitrobenzyl)sulfonium salts with tryptophan ethyl ester

Abstract Reaction of the water-soluble reporter group, dimethyl (2-hydroxy-5-nitrobenzyl)-sulfonium bromide, with l -tryptophan ethyl ester in aqueous solutions at pH 4.7 produces two diastereomeric monosubstitution products derived from alkylation of position 3 of the indole nucleus, followed by cyclization through the nucleophilic α-amino group. Comparison of these products with those formed during reaction of 2-hydroxy-5-nitrobenzyl bromide with tryptophan ethyl ester shows that they appear in the same characteristic ratio, 1.4:1. However, the monosubstitution products are formed in nearly quantitative yield when tryptophan ethyl ester is treated with an equimolar quantity of the sulfonium reagent, so that significantly less of the by-products are formed than in the case of the hydroxynitrobenzyl bromide. These findings confirm the applicability of dimethyl(2-hydroxy-5-nitrobenzyl) sulfonium salts in introducing the 2-hydroxy-5-nitrobenzyl reporter grouping into the indole nucleus of tryptophyl residues in proteins.

Phosphorescence studies of environmental heterogeneity for tryptophyl residues in proteins.

Phosphorescence studies of environmental heterogeneity for tryptophyl residues in proteins.

Riboflavin-tryptophan complex formation as a criterion for “buried” and “exposed” tryptophyl residues in proteins

Complex formation between riboflavin 5′-phosphate and tryptophyl residues is used as a criterion for distinguishing between “buried” and “exposed” tryptophyl residues in proteins. It is shown that native α-chymotrypsin (EC 3.4.4.5) has only one binding site for riboflavin 5′-phosphate; this suggests that one of the enzyme's seven tryptophyl residues is on the outside of the molecular or “exposed”, while the other six are “buried” and thus unavailable for the formation of complexes with riboflavin 5′-phosphate. This information was obtained from spectroscopic experiments in which two different concentration conditions were used: (a) tryptophyl residues in excess of riboflavin 5′-phosphate, and (b) riboflavin 5′-phosphate in excess of tryptophyl residues. Evaluation of the quantities ΔϵM, the molar absorption difference coefficient of the complex; K, the association constant of the complex; and n, the number of available tryptophyl residues, were derived from the data taken under these two conditions. The self-consistency of these data indicates that the presence of riboflavin 5′-phosphate per se does not affect the riboflavin-binding sites of α-chymotrypsin. Similar results were obtained from measurements of partially denatured α-chymotrypsin, which was shown to have an additional binding site for riboflavin 5′-phosphate. There is reasonable agreement between these experimental K and ΔϵM values, and published values pertaining to the complexes between riboflavin 5′-phosphate and tryptophan or serotonin. Also, the determined values of n, the number of riboflavin-5′-phosphate-binding sites of α-chymotrypsin, are consistent with other information about this protein. These observations suggest that riboflavin 5′-phosphate might be useful as a general tool for the location of “buried” and “exposed” tryptophyl residues of proteins.