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Ionization of the phenolic group of N-acetyltyrosynamide has been studied using second-derivative spectroscopy. At pH 12.5 the second-derivative spectrum of the model compound revealed the presence of derivative bands in a spectral region (between 250 and 270 nm) where interference coming from other ultraviolet-absorbing chromophores is negligible. One of these peaks (260-nm peak) has been employed for the determination of tyrosyl groups in mixtures containing the aromatic amino acids.

The effects of denaturants on the solvent accessibility to tyrosyl residues of apomyoglobin have been examined by means of second-derivative spectroscopy in the near-ultraviolet. Three apomyoglobins, i.e., sperm whale, horse, and tuna, were selected because of the different distribution of tyrosyl residues in their primary structure. The results are consistent with the occurrence of two independent consecutive events in the guanidine-induced denaturation pattern of apomyoglobin. The first event, which is responsible for the lack of the ability to bind the heme, has been proved to involve conformational changes in both the domains, i.e., segments 1-79 and 80-153, identified in the myoglobin molecule. However, the conformational changes are not of the same type. In fact, the solvent accessibility to tyrosine HC2 is increased probably because of a partial unfolding of the 80-153 domain. Conversely, the solvent accessibility to tyrosine B2 is decreased, thus indicating that a refolding occurs in some region of the N-terminal moiety (1-79 domain) of the molecule.

The heart ventricle myoglobin of bluefin tuna has been purified to an apparent homogeneity. The amino acid analysis has revealed only a limited number of substitutions between the myoglobins of yellowfin and bluefin tuna. The alpha-helix content of tuna myoglobin has been found considerably lower than that of mammalian myoglobin. No correlation has been discovered between the conformational stability and alpha-helix content. Denaturation experiments have shown that the whole structure of tuna myoglobin results from the interaction of two structural units which represent the product of independent folding processes. The structure of tuna myoglobin has been found more open and disorganized than that of sperm whale. This result has been related to the low content of electrostatic interactions and explained in terms of evolutive adaptations.

1. 1. The skeletal muscle myoglobin of the water buffalo ( Bos bubalus L.) has been purified by a rapid and simplified procedure. 2. 2. The amino acid composition of the myoglobin has been determined. 3. 3. The N -terminal amino acid is glycine. 4. 4. The results have been compared with other mammalian myoglobins, particularly that of the cow.

Inclusion in agarose gel significantly affects the conformational dynamics of native and acidic partly folded states of tuna apomyoglobin, a single tryptophan containing protein, as documented by frequency domain fluorometry investigations. The heterogeneity of the tryptophanyl emission decay increases on gel inclusion compared to that observed for free-in-solvent protein at both neutral and acidic pH, thus suggesting that the interconversion rate among conformational substates is somewhat reduced. The observation that this effect is much more pronounced for the partly folded state suggests that confined environments such as those existing in the living cells might favor the sequential folding process avoiding that structured intermediates rapidly convert into less structured ones.

The guanidine-induced unfolding of myoglobin as well as apomyoglobin has been found to involve the occurrence of at least a molecular intermediate observed at low denaturant concentrations, the molecular properties of which resemble those possessed by the acid-denatured form of the protein. The two partially unfolded forms show the same secondary structure and similar tryptophanyl fluorescence emission and polarization but exhibit marked differences in the tyrosine contributions to the near-ultraviolet circular dichroism and in the degree of solvent accessibility to tyrosyl residues. The molecular characterization of the two structural forms indicates that acids disorganize the 80–146 molecular domain identified in the myoglobin molecule to a great extent with respect to that induced by low guanidine concentration, whereas the structure of the 1–79 domain appears to be quite similar in the two molecular forms.

The perturbing effect of guanidium hydrochloride and pH on the molecular structure of water buffalo apomyoglobin has been investigated by circular dichroism in the far and near ultraviolet and by fluorescence. In the wavelength region between 320 and 260 nm the circular dichroic spectrum of the globin is highly structured and the contributions of the aromatic chromophores have been resolved. Buffalo apomyoglobin undergoes a structural transition at neutral pH which involves elements of the secondary and tertiary structure, as indicated by changes of dichroic activity of the peptide and aromatic chromophores and the fluorescence of the two tryptophanyl residues. The possibility of charge-transfer complex between indole and imidazole is discussed. A major structural transition with abrupt unfolding takes place in the pH region between 5.6 and 4.3. Below pH 4.3 the peptide helical residues, which survive the acid transition, appear to be resistent to further acidification to pH 2.0 while tryptophanyl emission is quenched and shifted to longer wavelengths. A structural transition occurs also in alkali above pH 10, which has been detected by the same techniques. The relationships between buffalo and sperm whale apomyoglobin are discussed.

The triplet state lifetime and the position of the O—O vibrational band of the tryptophanyl phosphorescence spectrum of two philogenetically distant apomyoglobins, i.e. tuna and sperm whale apomyoglobin, have been studied at different temperatures. The temperature dependence of the two phosphorescence parameters revealed that the tryptophanyl microenvironment possesses different dynamic characteristics in the two examined proteins. In particular, the tryptophanyl side chain of tuna apomyoglobin was found embedded into a less rigid microenvironmental cluster which possesses a higher degree of internal flexibility than that of sperm whale apomyoglobin.

Second derivative spectroscopy has been used to resolve the complex protein spectrum in the near-ultraviolet region and the contributions of the three aromatic chromophores have been evaluated. A method for the direct quantitative determination of phenylalanine and tryptophan in proteins has been carried out. Phenylalanine determination has been carried out in the spectral region between 250 and 265 nm, where there are no significant contributions from other aromatic chromophores. Tryptophan determination has been performed in the 290-295-nm region and the experimental values have been corrected for the presence of tyrosine. The results obtained on 10 highly purified proteins have been found in good agreement with those obtained from sequence analysis.

The tryptophanyl fluorescence decays of two myoglobins, i.e., sperm whale and tuna myoglobin, have been examined in the frequency domain with an apparatus which utilizes the harmonic content of a mode-locked laser. Data analysis was performed in terms of continuous distribution of lifetime having a Lorentzian shape. Data relative to sperm whale myoglobin, which possesses two tryptophanyl residues, i.e., Trp-A-5 and -A-12, provided a broad lifetime distribution including decay rates from a few picoseconds to about 10 ns. By contrast, the tryptophanyl lifetime distribution of tuna myoglobin, which contains only Trp-A-12, showed two well-separated and narrow Lorentzian components having centers at about 50 ps and 3.37 ns, respectively. In both cases, the χ2obtained from distribution analysis was lower than that provided by a fit using the sum of exponential components. The long-lived components present in the fluorescence decay of the two myoglobins do not correspond to any of those observed for the apoproteins at neutral pH. The tryptophanyl lifetime distribution of sperm whale apomyoglobin consists of two separated Lorentzian components centered at 2.25 and 5.4 ns, whereas that of tuna apomyoglobin consists of a single Lorentzian component, whose center is at 2.19 ns. Acidification of apomyoglobin to pH 3.5 produced a shift of the distribution centers toward longer lifetimes. The similarity between the values of the distribution centers of the long-lived components observed in the fluorescence decay of native myoglobins and those observed for apomyoglobins at acidic pH suggests that the long-lived component might arise from a conformational state (different from the native state) in which geometrical factors preclude energy transfer via Forster coupling from tryptophan to heme. This possibility has been further supported by the identification, in the steady-state emission spectrum of tuna myoglobin, of a component having an emission maximum similar to that observed at acidic pH, i.e., 335–337 nm. The difference between the tryptophanyl lifetime distributions of the two native holoproteins has been explained in terms of a higher degree of structural flexibility of tuna globin compared to that of sperm whale myoglobin. © 1989, American Chemical Society. All rights reserved.

A method of preparing homogeneous coated vesicles that eliminates the high sucrose concentrations heretofore used is presented. It is shown that sucrose at high concentrations dissociates the coat from coated vesicles. This reaction can explain the presence of empty coats observed with preparations obtained with high concentrations of sucrose. The protein and membrane lipid components have been analyzed by the intrinsic tryptophan and extrinsic diphenylhexatriene fluorescence, respectively. Analysis of mixtures of coated vesicles and baskets resolved the contributions of the two species to the fluorescence curves.

The extent of conformational substates of two apomyoglobins, i.e., sperm whale and tuna apomyoglobin, was investigated by examining the fluorescence decay in the frequency domain of the extrinsic fluorophore TNS [6-(p-toluidino)-2-naphthalenesulfonic acid] bound to the heme binding site. Data analysis was performed in terms of a continuous, unimodal lifetime distribution having a Lorentzian shape. The results were compared with those for the free fluorophore in an isotropic nonviscous solvent. The incorporation of TNS into the protein matrix resulted in a broadening of the lifetime distribution due to the microenvironmental heterogeneity generated by structural fluctuations. The larger width of lifetime distribution observed for TNS bound to tuna apomyoglobin was related to a more extended conformational space accessible to the fluorophore in this protein compared to sperm whale myoglobin. A temperature increase from 15 to 40 degrees C produced a further broadening of the lifetime distributions of TNS bound to both proteins. This result can be explained by assuming the existence of conformational substates at high energy content or separated by high energy barriers, which are not populated at low temperature. The overall picture emerging from the reported data is that the lifetime distributions of TNS bound to apomyoglobins are determined largely by the number of conformational substates accessible to the protein matrix and, to a lesser extent, by the interconversion rates among these states.

The reaction of yeast alcohol dehydrogenase with iodoacetate produces a loss of 90% of the enzymatic activity when 2.2–2.4 equivalents of carboxymethyl groups are incorporated. After CNBr cleavage of the 14C-carboxymethylated enzyme the more radioactive fragment was maleylated and digested with trypsin. A tryptic peptide, containing about 70% of initial radioactivity, was purified and sequenced. The cysteine found to be more reactive with iodoacetate in our experimental conditions is different from that found in previous works. It also appears to differ from the cysteine residues found to be reactive towards other thiol reagents.

Clathrin polymerizes to form characteristic coat structures (baskets) closely resembling those found on coated vesicles. Two sizes of baskets are formed from clathrin, depending on the purity of the preparation and on other factors. A protein of Mr 110 000 has been separated from clathrin by lysine-Sepharose chromatography which is needed for the formation of 150S baskets. In its absence, polymerization results in the larger size baskets, i.e., 300S. Addition of Ca2+ or Mg2+ stimulates 300S formation in the presence of the 110 000 protein.

— The second derivative spectra of tryptophan in water and in ethylene glycol at 22°C have been integrated in order to obtain the corresponding primitive functions. The integration was carried out by making use of Tchebychev polynomials. The results show that the integrated primitive functions do not correspond to the original absorption spectra of tryptophan in various solvents, but they reflect only the contributions of the 1Lb bands of the indolic chromophore. The identification of the electronic component, which generates the second derivative spectrum, was based on the solvent insensitivity of the derivative peaks. The comparison between the absorption spectra reported in this paper and those calculated for the 1Lb←1A electronic transition of indole confirmed the assumption that the derivation process eliminates the broad, although more intense, contributions coming from the 1La←1A electronic transition. Copyright © 1985, Wiley Blackwell. All rights reserved

The free energy of unfolding of horse myoglobin has been calculated from the denaturation pattern induced by guanidine hydrochloride as well as by acid. The delta GH2O, i.e., the value in the absence of denaturant obtained by using the two-state transition model, was found to be 25% lower than that determined from the acid denaturation pattern, i.e., 12.0 kcal/mol, although the extent of protein denaturation produced by acid was much lower. The amount of helical structure surviving the acid-induced conformational change was estimated to be 50% of that present in the native protein, and it could be destroyed only after exposure of myoglobin samples kept at pH 3.0 to concentrated guanidine. From the guanidine denaturation pattern at acidic pH, a further variation of free energy of unfolding of 5.5 kcal/mol could be calculated, thus indicating that the overall free energy of unfolding determined from the two consecutive processes corresponds to 17.5 kcal/mol. The discrepancy between the two sets of data, i.e., guanidine unfolding at neutral pH and acid unfolding followed by addition of denaturant, has been considered to depend on the general assumption that the guanidine unfolding of myoglobin is a two-state process in the transition region. According to the recent experimental evidence showing the occurrence of at least two molecular events during the guanidine unfolding of apomyoglobin [Colonna, G., Balestrieri, C., Bismuto, E., Servillo, L., & Irace, G. (1982) Biochemistry 21, 212-215], the guanidine denaturation pattern of myoglobin was analyzed in terms of two independent steps.(ABSTRACT TRUNCATED AT 250 WORDS)

The individual fluorescence of the two tryptophan residues (Trp-7 and Trp-14) of mammalian apomyoglobins has been resolved by comparing the fluorescence properties of these proteins to those of bluefin tuna apomyoglobin, which contains only Trp-14. The two tryptophan residues have been found to have different emission maxima, i.e., 321 for Trp-14 and 333 for Trp-7. The fluorescence of Trp-14 depends on the protonation of a sterically related histidyl residue in the pH range between 8.3 and 5.6, where no conformational change was detected. This residue has been identified as His-119. At pH 8.3 the quantum yield of Trp-7 is lower than that of Trp-14. An increase of the fluorescence intensity of Trp-7 occurs when the heme binding site of apomyoglobin is destroyed by acid or a low concentration of guanidine hydrochloride. An independent unfolding of the N-terminal district of the apomyoglobin molecular occurs on increasing the guanidine concentration. The two distinct structural transitions have been discussed in terms of two domains of tertiary structure.

The rates of oxidation of several goitrogens by lactoperoxidase and the rates of inactivation of lactoperoxidase by the same goitrogens have been measured. The influence of iodide on both reactions has also been evaluated. It has been shown by us that iodide acts catalytically in regulating lactoperoxidase activity at pH 8.8. The rate data have been analyzed by a computer program which solves the differential equations for the above mentioned reactions. From this computer analysis we have been able to obtain binding constants of the goitrogens and inactivation rate constants of lactoperoxidase. Iodide was shown to inhibit goitrogenic activity either by increasing the rate of drug oxidation or by reducing the rate of enzyme inactivation, or both, depending on the particular drug. Iodide had little or no effect on the goitrogen-binding constants. We have also shown that the relative rates of enzyme inactivation can be correlated with the potency of the goitrogen as an antithyroid drug.

The heme and cysteine microenvironments of bluefin tuna apomyoglobin have been investigated by examining the fluorescence properties of two extrinsic chromophores, i.e., ANS and 1,5-AEDANS. 1,5-AEDANS was covalently bound to the single cysteine residue found in the primary structure of tuna apomyoglobin. Recombination experiments with hemin showed that tuna apomyoglobin does not bind 1,5-AEDANS in the same binding site of the heme, although the fluorescence properties of the covalently bound 1,5-AEDANS strongly suggest that the dye is embedded in a rather nonpolar microenvironment. ANS was selected because of its ability to bind the apomyoglobin in the same nonpolar moiety of the heme. Acidification of apoMb--AEDANS to pH 3.0 produced an increase of 1,5-AEDANS fluorescence intensity and a shift of its emission maximum from 475 to 470 nm. In the same pH range apomyoglobin lost its ability to bind ANS. Two independent transitions were observed with increasing concentrations of guanidine. Low guanidine concentration (less than 1.0 M) unfolded the heme binding site as indicated by the disappearance of ANS fluorescence, whereas higher denaturant concentration was required to produce full normalization of 1,5-AEDANS emission spectrum.

The effects of the antithyroid goitrogens, methylthiouracil and methylmercaptoimidazole, on the oxidation of N-acetyltyrosylamide at pH 8.8 by lactoperoxidase have been evaluated in the presence and the absence of iodide for the purpose of elucidating the effects of iodide. At pH 8.8, iodine is not oxidized. In the absence of iodide, the two antithyroid drugs inactivate lactoperoxidase by a second order process. When iodide is added before methylthiouracil or methylmercaptoimidazole, enzyme inactivation does not occur as rapidly and both goitrogens are readily oxidized. The kinetics of the oxidation reactions have been analyzed in order to obtain the equilibrium constant of the iodide . lactoperoxidase complex. Essentially the same iodide dissociation constant, i.e. 2 x 10(-5) M, was found by studying its effects on the kinetics of oxidation of the two antithyroid drugs. A large difference absorption spectrum is observed in the Soret region between native lactoperoxidase and lactoperoxidase inactivated by methylthiouracil.

Dynamic aspects of the heme-binding site of myoglobins derived from two phylogenetically distant species, namely sperm whale and bluefin tuna, have been investigated by studying steady-state and time-resolved emission properties of 2-p-toluidinyl-6-naphthalene sulfonic acid (TNS) apomyoglobin conjugates. Multi-frequency phase and modulation fluorometry data indicate that charge movements occur in the fluorophore environment during the excited state lifetime in the sperm whale myoglobin system. In the case of the bluefin tuna myoglobin TNS adduct these movements were not detected, indicating that the relaxation processes differ in the two types of myoglobins.

Proteins exhibit, even in their native state, a large number of conformations differing in small details (substates). The fluorescence lifetime of tryptophanyl residues can reflect the microenvironmental characteristics of these subconformations. We have analyzed the lifetime distribution of the unique indole residue of tuna apomyoglobin (Trp A-12) during the unfolding induced by temperature or guanidine hydrochloride. The results show that the increase of the temperature from 10 to 30 °C causes a sharpening of the lifetime distribution. This is mainly due to the higher rate of interconversion among the conformational substates in the native state. A further temperature increase produces partially or fully unfolded states, resulting in a broadening of the tryptophanyl lifetime distribution. The data relative to the guanidine-induced unfolding show a sigmoidal increase of the distribution width, which is due to the transition of the protein structure from the native to the random-coiled state. The broadening of the lifetime distribution indicates that, even in the fully unfolded protein, the lifetime of the tryptophanyl residues is influenced by the protein matrix, which generates very heterogeneous microenvironments. © 1988, American Chemical Society. All rights reserved.

1. 1. A method for the purification of alkaline phosphatase (orthophosphoric monoester phosphophydrolases, EC 3.1.3.1) from beef brain has been described. 2. 2. Two alkaline phosphatase fractions have been separated by means of DEAE-cellulose chromatography. The main fraction was homogeneous on polyacrylamide gel electrophoresis. 3. 3. The molecular weight of the enzyme determined by gel filtration chromatography has been estimated to be about 180,000. The Michaelis constant and optimum pH for p-nitrophenyl phosphate have been determined. 4. 4. The results have been compared with those of another recently reported alkaline phosphatase isolated from the same source. The dissimilarity of two enzymes is described and discussed.

The primary structure of fibrinopeptides A and B from buffaloes breeding ing in Italy has been determined with a view to establishing whether this animal is an autochthonous species or is the result of recent radiation mutation. Some differences exist at the morphologic and physiologic levels between the Indian buffalo and that breeding in Italy, but they do not allow a clear evolutionary line to be traced between these species. The amino acid sequences, if compared with homologous sequences of the Indian buffalo, show one difference in the most variable region of fibrinopeptides A, in particular the substitution of a serine residue by a glycine residue in position 8. This difference supports the hypothesis of the autochthonous origin of the Italian buffalo.

The accessibility of the heme binding site of two apomyoglobins, i.e. tuna and sperm whale apomyoglobin, has been evaluated by quenching the fluorescence of their ANS-conjugates. The quenching pattern obtained by using charged and uncharged quenchers revealed that the heme pocket of tuna apomyoglobin is more accessible than that of sperm whale. Moreover, a larger number of positively charged groups is present in the heme pocket of tuna apomyoglobin as indicated by comparing the extent of quenching produced by iodide and cesium ion. The relaxation time of ANS bound to tuna apomyoglobin is lower than that of the same chromophore bound to sperm whale globin thus indicating that there is some localized flexibility in the tuna globin.

The mutual interference between the second-derivative bands of tyrosine and tryptophan in proteins has been evaluated in terms of the ratio r between two peak-to-peak distances. The r values have been found to be well related, although not linearly, to the tyrosine/tryptophan ratio in both model compound mixtures and proteins. A method for the simultaneous determination of the two major protein chromophores at neutral pH has been developed.

The free energy of unfolding of several myoglobins from different animal species has been determined from their denaturation pattern by using the ligand binding model. The results indicate that no simple correlation exists between the free energy of unfolding of myoglobin and the basal metabolic rate of the animal species from which the myoglobin was isolated.

The nature of the molecular event that apomyoglobin undergoes at predenaturational concentrations of guanidine has been investigated by means of steady-state and multifrequency phase and modulation fluorometry. The results have been compared to those observed for liver alcohol dehydrogenase. From these studies has been hypothesized a different susceptibility of the distinct elements of secondary, super-secondary, and tertiary structure towards the denaturing action of guanidine at predenaturational concentrations.

The effects of heme removal on the molecular structure of tuna and sperm whale myoglobin have been investigated by comparing the solvent accessibility to the heme pocket of the two proteins with that of the corresponding apoproteins. Although the heme microenvironment of tuna myoglobin is more polar than that of sperm whale myoglobin, the accessibility of solvent to heme is identical in the two proteins as revealed by thermal perturbation of Soret absorption. The removal of heme produces loss of helical folding and increase of solvent accessibility but the effects are rather different for the two proteins. More precisely, the loss of helical structure upon heme removal is 50% for tuna myoglobin and 15% for sperm whale myoglobin; moreover, the solvent accessibility of the heme pocket of tuna apomyoglobin is 2-3-fold greater than that of sperm whale apomyoglobin. These results have been explained in terms of the lack of helical folding in segment D, the structural organization of which may have a relevant effect in regulating the accessibility of ligands to the heme. The effects produced by charged quenchers reveal that the ligand path from the surface of the molecule to the ion atom of the heme involves a positively charged residue which may reasonably be identified as Arg-45 (sperm whale myoglobin) or Lys-41 (tuna myoglobin) on the basis of recent X-ray crystallographic information.

The circular dichroic activity of tuna myoglobin in the far ultraviolet has been found to be lower than that of mammalian myoglobin, thus indicating a lower content of alpha-helix. Fluorescence and absorption studies have indicated that the structure of the N-terminal region of the molecule is essentially the same in all the examined apomyoglobins, whereas differences have been observed in the heme microenvironment. The prediction of secondary structure has revealed that the alpha-helical segments of tuna myoglobin, especially those involved in the formation of the heme pocket, are shorter than those of sperm whale myoglobin.

Indolamine N-methyltransferase (INMT) has been purified to an apparent homogeneity from rabbit lung, and some of its catalytic and physicochemical properties have been examined. The enzyme is a monomeric protein with a molecular weight of 31,500 +/- 1000, a molecular Stokes radius of 21.5 A, and a diffusion coefficient of 8.7 X 10(-7) cm2/s. The frictional ratio of the native enzyme (1.05) suggests that the shape of the molecule is nearly spherical. Denaturation experiments performed with increasing concentrations of guanidine hydrochloride (Gdn-HCl) at neural pH indicated that the active site of the enzyme was destroyed by a structural rearrangement of the protein molecule without large change in its size and shape. The final state reached in 6.0 M Gdn . HCl seemed to correspond to a disulfide cross-linked randomly coiled polypeptide. Full normalization of the fluorescent parameter was attained only in the presence of 0.1 M beta-mercaptoethanol. A structural rearrangement has been observed upon acidification of INMT from pH 7.0 to pH 2.0. At pH 4.5, most of the peptide backbone appeared to be unorganized, but further acidification to pH 2.0 produced a reorganization of protein structure which became able to bind 8-anilino-1-naphthalenesulfonate. The data support the hypothesis that the enzyme structure results from the close package of organized regions joined by structureless segments.

Mammalian myoglobins contain two tryptophanyl residues at the invariant positions A-5 (W7) and A-12 (W14) in the N-terminal region (A helix) of the protein molecule. To determine the contribution of each tryptophanyl residue to the structure and stability of myoglobin, recombinant proteins with single indole residue, i.e., W7 or W14, were obtained by site-directed mutagenesis. The mutant proteins, expressed in Escherichia coli, were found correctly folded, the far ultraviolet circular dichroism of both mutants as well as the Soret absorption being superimposed to that of wild type protein. The removal of the prosthetic group from mutant proteins determined a loss of helical content much larger than that observed in the case of wild type myoglobin. These results suggest that tryptophanyl residues can play a crucial role on globin folding and structure.

A molecular dynamics simulation approach has been utilized to understand the unusual fluorescence emission decay observed for beta-glycosidase from the hyperthermophilic bacterium Solfolobus sulfotaricus (Sbeta gly), a tetrameric enzyme containing 17 tryptophanyl residues for each subunit. The tryptophanyl emission decay of Sbeta gly results from a bimodal distribution of fluorescence lifetimes with a short-lived component centered at 2.5 ns and a long-lived one at 7.4 ns (Bismuto E, Nucci R, Rossi M, Irace G, 1999, Proteins 27:71-79). From the examination of the trajectories of the side chains capable of causing intramolecular quenching for each tryptophan microenvironment and using a modified Stern-Volmer model for the emission quenching processes, we calculated the fluorescence lifetime for each tryptophanyl residue of Sbeta gly at two different temperatures, i.e., 300 and 365 K. The highest temperature was chosen because in this condition Sbeta gly evidences a maximum in its catalytic activity and is stable for a very long time. The calculated lifetime distributions overlap those experimentally determined. Moreover, the majority of trytptophanyl residues having longer lifetimes correspond to those originally identified by inspection of the crystallographic structure. The tryptophanyl lifetimes appear to be a complex function of several variables, such as microenvironment viscosity, solvent accessibility, the chemical structure of quencher side chains, and side-chain dynamics. The lifetime calculation by MD simulation can be used to validate a predicted structure by comparing the theoretical data with the experimental fluorescence decay results.