Changes in the exposure of tryptophyl and tyrosyl residues in thrombin due to diisopropylphosphorofluoridate and benzamidine inhibitions.

Chemical modifications of tyrosine and tryptophan residues in diisopropylphosphoryl-thrombin (DIP-thrombin) and benzamidine-inhibited thrombin (BA-thrombin) by N-acetylimidazole and hydrogen peroxide-dioxane mixture indicate the burial of two tyrosyl and two tryptophyl residues relative to the active enzyme. During inhibition the circular dichroism spectra in the peptide-absorbing region is apparently unchanged while small detectable changes are observed in the aromatic region. It is concluded that tryptophan and tyrosine residues are part of the structural features of the active center of thrombin but they do not play active roles in the catalytic process.     

Ring-Toss: Capping highly exposed tyrosyl or tryptophyl residues in proteins with beta-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.     

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 β-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 β-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 β-cyclodextrin, although they reacted positively to dimethyl sulfoxide solvent perturbation. These findings indicate that β-cyclodextrin perturbation provides a convenient way to detect highly exposed tyrosyls or tryptophyls in proteins. Furthermore, we evaluated the ability of β-cyclodextrin to inhibit the interaction of turkey ovomucoid third domain variants with different P₁ residues. The results showed that the presence of β-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, β-cyclodextrin may be used to selectively modulate the interaction between proteinase inhibitors and their cognate enzymes.     

Fluorimetric studies of tryptophyl exposure in concanavalin A.

Studies of the iodide ion quenching of the intrinsic fluorescence of Concanavalin A indicate that 50% of the tryptophyl fluorescence originates from exposed residues. This agrees with the X-ray crystallographic determination that two of the four tryptophan residues in a Concanavalin A monomer are on the surface. Previous studies have indicated that conformational changes induced by sugar binding alter the environment of aromatic residues. The present investigation finds that neither the specific binding of alpha-methyl-D-mannoside nor alteration of the Concanavalin A quaternary structure changes the number or accessibility of the solvent-exposed tryptophan residues. It therefore appears that the major conformational transitions in Concanavalin A do not affect steric access to the surface tryptophans and the effects previously observed may be ascribed to structurally internal tryptophan residues.     

Fluorimetric and spectrophotometric studies of DPN-linked isocitrate dehydrogenase from bovine heart. Properties of tyrosyl and tryptophyl residues.

The emission maximum of DPN-linked isocitrate dehydrogenase in pH 7.07 buffer is shifted from 317 to 324 nm and fluorescence intensity is decreased when the excitation wave-length is varied from 270 to 290 nm; in 0.2 M KOH, where the fluorescence of tyrosyl residues is almost completely quenched, a further substantial decline in quantum yield of protein fluorescence and a red shift of the emission peak to 339 nm occur. The latter should be due mainly to tryptophyl residues. The enzyme contains 9.4 tyrosyl residues per subunit of molecular weight 42,000 determined spectrophotometrically (295 nm) at pH 13, in good agreement with a tyrosine content of 9.7 by amino acid analysis. No more than 1.1 tyrosyl residues per subunit can be detected up to pH 10.6 at 7 degrees upon prolonged incubation. The increase in absorption at 295 nm with increasing pH is related to loss of enzyme activity and results in a red shift of the emission maximum, and decreased fluorescence intensity. Treatment of the enzyme in a Li+-containing buffer at pH 7.5 with an excess of N-acetylimidazole results in (a) modification of 1.1 tyrosyl residues per subunit, (b) a 30% decrease in enzyme activity, (c) a 6-nm red shift in emission maximum, and (d) a decrease in fluorescence intensity. Manganous DL-isocitrate (1.06 mM) prevents the acetylation of the enzyme. Deacetylation of the O-acetylated enzyme by hydroxylamine completely restores the enzyme activity and reverses the spectral changes. The acetylation studies indicate that the reactive tyrosyl residue does not participate directly in catalysis but may be involved in maintaining the proper conformation of the active enzyme center. A net of 1 of the 2 tryptophyl residues per subunit is perturbed immediately by a number of solvents. This perturbation is not affected by manganous isocitrate, whereas exposure of tyrosyl residues occurs only with time and is prevented by the substrate. The perturbation of the tryptophyl residue is accompanied by a red shift of the fluorescence emission maximum. The more exposed tryptophyl residue may contribute to the energy transfer from protein to nucleotides since the quenching of protein fluorescence upon binding of DPN+, DPNH, or ADP by enzyme results in a blue shift of the emission maximum. Manganous DL-isocitrate (1.06 mM) quenches protein fluorescence by 16% without a shift in emission peak and does not affect the relative extent of fluorescence quenching induced by the nucleotides.     

The states of tyrosyl and tryptophyl residues in a protein proteinase inhibitor (Streptomyces subtilisin inhibitor

The states of tyrosyl and tryptophyl residues of a dimeric protein proteinase inhibitor, Streptomyces subtilisin inhibitor (Sato, S & Murao, S. (1973), Agric. Biol. Chem. 37, 1067) were studies by solvent perturbation difference spectroscopy with methanol, ethylene glycol, polyethylene glycol, and deuterium oxide as perturbants, and by spectrophotometric titration at alkaline pH. It appeared that all three tyrosyl residues per monomer of the inhibitor were exposed on the surface of the molecule, and their apparent pK values were estimated separately to be 9.58, 11.10, and 12.42. The single tryptophyl residue per monomer of the inhibitor appeared to be partially buried in the protein molecule.     

Measurement and calibration of peptide group hydrogen-deuterium exchange by ultraviolet spectrophotometry.

An exceedingly simple and convenient method is described for measuring the hydrogen-deuterium exchange behavior of peptide bond-containing molecules by ultraviolet spectrophotometry. The exchange reaction is initiated by diluting a sample from H₂O into D₂O, or the reverse, and can be followed by an easily observable optical density change in the region of peptide absorbance. The method, unlike infrared and magnetic resonance approaches, requires only small amounts of material and, unlike the tritium-Sephadex method, is not restricted to the study of large molecules. Calibrations are provided for exchange rate as a function of pD and temperature and for the change in absorbance per mole peptide group. With this information, the exchange curve to be expected for any peptide group exposed to solvent can be predicted. Comparison with the measured data can then identify peptide-group hydrogen bonding and can also give a measure of the stability of the hydrogen-bonded structure.