Interaction of 'toxic' and 'immunogenic' A-gliadin peptides with a membrane-mimetic environment.

Celiac disease (CD) is characterized by abnormally high concentrations of certain peptides in the small bowel. These peptides can be grouped in 'toxic' and 'immunogenic' classes, which elicit an innate immune response and an HLA-mediated adaptive response, respectively. It is not clear on which molecular mechanisms responses to these different classes are based, but the 31-43 (P31-43) and the 56-68 (P56-68) A-gliadin fragments are usually adopted as sequence representatives of toxic and immunogenic peptides, respectively. Here we report fluorescence experiments aiming to mimic the interaction of these peptides with the cell membrane surface by using sodium dodecyl sulphate (SDS) as a membrane-mimetic medium. We show that P31-43 is able to bind SDS micelles in a way that resembles mixed micelle formation. On the other hand, no binding at all could be detected for P56-68. This different behaviour could be related to the paracellular or transcellular route through which gluten peptides may cross the intestinal epithelium, and open new insights into the pathogenetic mechanisms of CD.

The effect of molecular confinement on the conformational dynamics of the native and partly folded state of apomyoglobin.

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.

Role of tryptophanyl residues in driving myoglobin folding.

Mammalian myoglobins contain two tryptophanyl residues at the invariant positions 7 (A-5) and 14 (A-12) in the N-terminal region (A helix) of the protein molecule. The simultaneous substitution of both tryptophanyl residues causes an incorrect folding with subsequent loss of heme binding. The introduction of a indolic residue in different molecular regions, i.e. G, E, and C helix resulted in a not correctly folded protein, suggesting that the tryptophanyl residues are strong structural determinants.

Tryptophanyl fluorescence lifetime distribution of hyperthermophilic beta-glycosidase from molecular dynamics simulation: a comparison with the experimental data.

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.

The effect of tryptophanyl substitution on folding and structure of myoglobin.

Mammalian myoglobins contain two tryptophanyl residues at the invariant positions 7 (A-5) and 14 (A-12) in the N-terminal region (A helix) of the protein molecule. The crucial role of tryptophanyl residues has been investigated by site-directed mutagenesis and molecular dynamics simulation. The apomyoglobin mutants with a double W-->F substitution were found to be not correctly folded and therefore not expressed as holoprotein. The introduction of a tyrosyl residue at position 7, that is, W7YW14F, resulted in the expression of a correctly folded myoglobin. Not correctly folded apomyoglobins were found with the following mutants: W7FW14Y, W7EW14F, W7FW14E, W7KW14F, W7FW14K. Moreover, in all these cases, very low levels of expression were observed. The acid-induced denaturation curves of wild-type and folded mutant W7YW14F, obtained following the fluorescence variation of the extrinsic fluorophore 1-anilino-8-naphthalenesulfonate, revealed that the stability of the native state of mutant apoprotein is decreased, thus indicating that the replacement W-->Y in position 7 is able to restore a correct folding but not the same stability. Molecular dynamics simulation indicated that both tryptophans are involved in forming favorable, specific tertiary interactions in the native apomyoglobin structure. The lack of some of these interactions caused by tryptophanyl replacement affects the overall protein structure and may provide an explanation for the observed stability decrease. In the case of the double W-->F substitution, the simulated structure shows conclusively the domain formed by helices A, G and H to be not correctly folded. This effect is attenuated if at least one of the two residues is conserved or a tyrosyl residue replaces W7.

Tryptophanyl contributions to apomyoglobin fluorescence resolved by site-directed mutagenesis.

The individual emission properties of the two tryptophanyl residues of sperm-whale apomyoglobin have been resolved by examining the fluorescence variations induced by denaturants, i.e., acid and guanidine, on apomyoglobin mutants W7F and W14F. The fluorescence changes have been correlated to the conformational transitions undergone by apomyoglobin on increasing denaturant concentration. The results indicate that the fluorescence decrease, observed for sperm-whale apomyoglobin on going from pH 8.0 to pH 6.0, cannot be ascribed to the formation of a charge transfer complex between a nearby histidine residue and W14 as reported in earlier papers but rather to minor structural changes affecting the microenvironments of both residues. The formation of the acidic partly folded state around pH 4.0 determines an increase of the fluorescence yield and a small red shift (5 nm) of W7 due to removal of sterically interacting K79, which is able to attenuate the emission of this residue in the native state. The fluorescence intensity of the other residue, i.e., W14, is not affected by the acidic transition. Guanidine denaturation experiments revealed an increase of fluorescence yield of W14 upon the intermediate formation, whereas the fluorescence of the other residue remained constant. The results suggest that the unfolding pathway may be different depending on the chemical nature of the denaturant used.

Structural and dynamic aspects of beta-glycosidase from mesophilic and thermophilic bacteria by multitryptophanyl emission decay studies.

The tryptophanyl emission decay of beta-glycosidase from the extremophilic archaeon Sulfolobus solfataricus (Sbetagly) has been investigated by frequency domain fluorometry. The data were analyzed in terms of sum of discrete lifetimes as well as in terms of quasi- continuous lifetime distributions of different shape. At neutral pH the emission decay is characterized by two components: a long-lived component, centered at 7.4 ns, and a short one at 2.7 ns, irrespective of the decay scheme used for the interpretation of the experimental results. The effects of an irreversible inhibitor, that is, cyclophellitol, and that of a powerful denaturant such as guanidinium hydrochloride on the dynamics of Sbetagly has been investigated by observing the changes induced in the two components of the tryptophanyl emission decay. The addition of cyclophellitol to native Sbetagly reduces the contribution of the short-lived component but does not affect the long-lived one. Increasing concentrations of guanidinium hydrochloride differently affect the contributions of the two emission components. Higher concentrations were required to unfold the molecular regions containing the long-lived indolic fluorophores. These results indicate that the long-lived contribution arises from tryptophanyl residues deeply clustered in the interior of the protein matrix, whereas the short-lived one includes residues located in less rigid and more solvent accessible regions, some of which might be located in functionally important parts of protein. The knowledge of the crystallographic structure of Sbetagly allowed us to evaluate some average parameters for each tryptophanyl microenvironment in the Sbetagly such as hydrophobicity, structural flexibility, and ability of side chains to act as fluorescence quenchers. These results permitted to divide the tryptophanyl fluorescence of Sbetagly in the contribution of two emitting groups: one consisting of eight closely clustered tryptophans, that is, Trp 33, 36, 60, 84, 151 174, 425, and 433, responsible for the long-lived emission component and the other one, composed of nine tryptophans nearer to the subunit surface, that is, Trp 12, 156, 192, 287, 288, 316, 361, 376, 455, associable to the short-lived emission component. Finally, the examination of the tryptophanyl emission decay of the mesophilic beta-galactosidase from Escherichia coli (Cbetagal) and the Arrhenius analysis of its dependence on temperature indicated that the tryptophanyl environments of the mesophilic enzyme are rather homogeneous in consequence of a larger protein dynamics.

Apomyoglobin folding intermediates characterized by the hydrophobic fluorescent probe 8-anilino-1-naphthalene sulfonate.

Folding apomyoglobin intermediates were investigated by optical techniques including steady-state fluorescence, frequency domain fluorometry, and absorption spectroscopy. The investigated chromophores were the aromatic residues, i.e., tyrosyl and tryptophanyl residues, and the extrinsic probe (8-anilino-1-naphthalenesulfonate, ANS) which is particularly useful for studying partly structured forms appearing in the early stage of protein folding. The emission decay of the extrinsic probe as well as resonance energy transfer from tryptophanyl residues to ANS permitted to identify and characterize partly folded forms obtained under different experimental conditions. The results indicate that the intermediates so far detected (I-1 and I-2 states) are distinct structural states. The differences concern the solvent accessibility to the aromatic side chains and the conformational dynamics of the protein region forming the binding site for the extrinsic fluorophore.

Near-ultraviolet circular dichroic activity of apomyoglobin: resolution of the individual tryptophanyl contributions by site-directed mutagenesis.

The individual tryptophanyl contributions to the near-ultraviolet circular dichroic activity of apomyoglobin in its native conformation have been resolved by studying recombinant proteins with single tryptophanyl substitutions. Site-directed mutagenesis of sperm whale apomyoglobin was performed in order to obtain proteins containing only Trp A-5 or Trp A-12. These amino acid substitutions have very little effect on the overall globin fold as indicated by comparing the spectroscopic properties of the mutants with those of the wild type protein. The circular dichroism spectra of the two apomyoglobin mutants in the near ultraviolet were found to be significantly different, both indole residues having significant activity but of opposite sign. In particular, Trp A-5 shows the presence of a main positive peak centered near 294-295 nm with a marked shoulder at 285 nm, ascribed to the 1LB transition. The spectrum of the mutant protein containing only Trp A-12 shows a large negative contribution with a minimum near 283 nm and a marked shoulder at 293 nm. The broadness of the negative contribution exhibited by Trp A-12 suggests that it may originate mainly from the 1LA transition.

Single tryptophanyl substitutions affect the structure of apomyoglobin.

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.

Multitryptophan-fluorescence-emission decay of beta-glycosidase from the extremely thermophilic archaeon Sulfolobus solfataricus.

The emission decay of intrinsic fluorescence of the extremely thermophilic beta-glycosidase from Sulfolobus solfataricus has been investigated as functions of temperature and of iodide-quencher concentration by frequency-domain fluorometry. This protein contains 68 tryptophans and provides a matrix for correlation of the average spectroscopic behaviour with solvent exposure and local dynamics. At each temperature, the emission is very heterogeneous and interpretable in terms of quasicontinuous bimodal distribution of fluorescence lifetimes. We associate the component of the bimodal distribution to two distinct classes of tryptophanyl residues that differ in microenvironmental characteristics. Temperature and quenching experiments show that the long-lived component includes tryptophanyl residues located in buried regions with high rigidity; the short distributional component corresponds to tryptophans embedded in more flexible and exposed regions. This proposal has been confirmed by examination of the crystallographic structure. The data suggest that, at least for this protein, there is a good correlation between residue exposure and lifetime distributional components. The conformational dynamics of the two classes of tryptophanyl residues is affected differently by temperature, suggesting that the protein regions in which they are located give different contributions to enzyme properties, such as flexibility, stability and function.

Perturbation of conformational dynamics, enzymatic activity, and thermostability of beta-glycosidase from archaeon Sulfolobus solfataricus by pH and sodium dodecyl sulfate detergent.

The conformational dynamics of beta-glycosidase from Sulfolobus solfataricus was investigated by following the emission decay arising from the large number of tryptophanyl residues that are homogeneously dispersed in the primary structure. The fluorescence emission is characterized by a bimodal lifetime distribution, suggesting that the enzyme structure contains rigid and flexible regions, properly located in the macromolecule. The enzyme activity and thermostability appear to be related to the dynamic properties of these regions as evidenced by perturbation studies of the enzyme structure at alkaline pH and by addition of detergents such as SDS. The pH increase affects the protein dynamics with a remarkable loss of thermal stability and activity; these changes occur without any significant variation in the secondary structure as revealed by far-UV dichroic measurements. In the presence of 0.02% (w/v) SDS at alkaline pH, the enzymatic activity and thermostability are recovered. Under these conditions, the conformational dynamics appear to be similar to that evidenced at neutral pH. Further increases in SDS concentration, at alkaline pH, render the activity and thermostability of beta-glycosidase similar to those observed in the absence of detergent.

Pressure-induced perturbation of ANS-apomyoglobin complex: frequency domain fluorescence studies on native and acidic compact states.

The pressure dependence of the flexibility of the 8-anilino-1-naphthalene sulfonate (ANS)-apomyoglobin complex was investigated in the range between atmospheric pressure and 2.4 kbar by frequency domain fluorometry. We examined two structural states: native and acidic compact. The conformational dynamics of the ANS-apomyoglobin complex were deduced by studying the emission decay of ANS, which can form a noncovalent complex with the apoprotein in both the native and the acidic compact forms. Because the free fluorophore has a very short lifetime (less than 75 ps), its contribution can be separated from the long-lived emission. The latter arises from ANS molecules bound to the protein and provides information on the structural and dynamic characteristics of the macromolecule. The fluorescence emission decay of the ANS-apomyoglobin complex at neutral pH has a broad fluorescence lifetime distribution (width at half-maximum = 4.1 ns). The small changes in the fluorescence distribution parameters that occur with changes in pressure indicate that the ANS-apomyoglobin complex at neutral pH holds its compactness even at 2.4 kbar. A small contraction of molecular volume has been detected at low pressure, followed by a slight swelling with an increase in flexibility at higher pressures. The heterogeneity of ANS fluorescence in the acidic compact state of apomyoglobin is even greater than that in the native form (distribution width = 10 ns); moreover, the acidic compact state appears more expanded and accessible to solvent molecules than the native state, as suggested by the distribution center, which is 11 ns for the former and 19 ns for the latter. The lifetime distribution center remains constant with increasing pressure, which suggests that no other binding site is formed at high pressure.

Pressure-induced perturbation of apomyoglobin structure: fluorescence studies on native and acidic compact forms.

The nature of the structural changes that apomyoglobin undergoes when subjected to hydrostatic pressure, ranging from atmospheric pressure to 2.4 kbar, has been investigated by steady-state fluorescence and frequency domain fluorometry. In particular, we have examined the intrinsic tryptophanyl emission and that of the extrinsic probe 1-anilino-8-naphthalenesulfonate (ANS) bound to apomyoglobin at neutral pH, as well as at strongly acidic high-salt conditions. Apomyoglobin at neutral pH undergoes a pressure-induced structural transition, which causes the disorganization of the heme binding region with a consequent ANS dissociation; a concomitant increase in solvent accessibility to the N-terminus of the macromolecule in which tryptophans are located is also observed. At 2.4 kbar, the tryptophanyl emission is not coincident with that of a fully solvent exposed residue, thus suggesting that the N-terminal region of the apomyoglobin molecule retains elements of organized structure. The spectroscopic properties of the structural state attained at 2.4 kbar and neutral pH are different from those of the acidic compact state. The acidic compact state of apomyoglobin undergoes a pressure-induced structural change that brings the tryptophanyl residues in contact with the solvent, but does not affect the ability to bind ANS.

Unfolding pathway of apomyoglobin. Simultaneous characterization of acidic conformational states by frequency domain fluorometry.

The dynamic properties of the conformational states co-existing during the acid-induced unfolding of tuna apomyoglobin, a single tryptophan-containing protein, have been investigated simultaneously by frequency domain fluorometry. In the transition region, in the absence of salt, the tryptophanyl fluorescence emission arises from a bimodal lifetime distribution. The pH decrease causes a marked broadening of the short-lived distribution component whereas the other component, i.e. the long-lived one, remains unchanged and represented by a very narrow lifetime distribution whose width is similar to that of the native protein. The broadening of the short-lived distribution component observed on lowering the pH indicated that this component arises from fully unfolded molecules. This was further corroborated by acrylamide quenching studies at acidic pH. The collisional quenching rate constant of the short-lived distribution component, i.e. 8.9 x 10(9) M-1s-1, was found to be similar to that observed for a fully exposed residue. The long-lived distribution component was characterized by a lower collisional quenching rate constant, i.e. 2.3 x 10(9) M-1s-1. This value if compared to that determined for the native apoprotein at neutral pH, i.e. 4.0 x 10(8) M-1s-1, indicates that the native-like structure surviving the acid-induced transition possesses a large molecular flexibility.

Resolution of the individual tryptophanyl contributions to the near-ultraviolet dichroic activity of apomyoglobin.

The individual tryptophanyl contributions to the near-ultraviolet dichroic activity of apomyoglobin in its native conformation have been resolved. This was accomplished by comparing the spectra of two classes of apomyoglobin with different aromatic residue contents and observing the effect of a specific modification of indole residues. The circular dichroism (CD) spectra of apomyoglobins containing two tryptophanyl residues, i.e. Trp A-5 and A-12, show the presence of a positive peak centered at 292 nm, attributable to indolic chromophore, which is missing in the CD spectrum of tuna apomyoglobin possessing only Trp A-12. Moreover, the specific modification of Trp A-5 by 2-hydroxy-5-nitrobenzyl bromide is shown by the lack of the 292 nm peak and the appearance of a positive band at longer wavelength. The pH dependence of the position of this band suggests that it arises from the 2-hydroxy-5-nitrobenzyl moiety. The results suggest that Trp A-12 does not substantially contribute to the optical activity in the near ultraviolet.

Solvent and thermal denaturation of the acidic compact state of apomyoglobin.

The stability of the acidic compact state of apomyoglobin toward the denaturant action of guanidinium hydrochloride and temperature was studied by examining the effects induced on the intrinsic tryptophanyl fluorescence and that of the adduct formed with 1,8-anilinonaphthalenesulfonate (ANS). The results indicated that the disorganization of tryptophanyl environments is caused by a cooperative discrete molecular transition, thus contrasting the assumption that the acidic compact form of apomyoglobin might be a molten globule state. The unfolding of the ANS binding regions was found to involve, at least, two stages over a wide range of denaturant concentrations.

Structure and dynamics of the acidic compact state of apomyoglobin by frequency-domain fluorometry.

The conformational dynamic properties of tuna apomyoglobin, a single tryptophan-containing protein, in the acidic compact state, as well as in the native and in the fully unfolded state, have been explored by frequency-domain fluorometry. Apomyoglobin at acidic pH in the presence of high salt concentration displays bimodal tryptophanyl lifetime distributions which may be related to the simultaneous presence of different populations of structural states (compact and fully unfolded states). The tryptophanyl anisotropy decay indicated that the acidic compact state displays at least two rotational correlational times, suggesting that this state possesses a complex geometrical organization. 1-Anilino-8-naphthalene sulfonate (ANS), bound both to native and compact protein forms, shows broad unimodal lifetime distributions. The small time dependence of the ANS emission spectra indicated that the solvent dipolar reorganization are either absent or they occur on a time scale much shorter than the lifetime of the excited ANS molecule bound to apomyoglobin. The anisotropy decay data relative to the extrinsic fluorophore (ANS) are consistent with the presence of a single rotational correlation time for both native (12.1 ns) and compact (6.2 ns) states.

Folding and dynamics of melittin in reversed micelles.

The main structural characteristics and the dynamic properties of melittin bound to the internal surface of reversed micelles, formed by sodium bis(2-ethyl-1-exyl)sulfosuccinate (AOT) in isooctane, were investigated by several spectroscopic techniques. Melittin has been found associated to reversed AOT micelles in a single state, thus indicating that this system behaves differently with respect to phospholipid vesicles where at least two forms of lipid associated melittin are observed. The dynamic properties of melittin in reversed AOT micelles at different water contents were examined by frequency domain fluorometry. The whole emission decay was analyzed in terms of lifetime distribution having a Lorentzian shape. The results indicated that the binding of melittin to inverted micelles determines an increase of emission heterogeneity compared to that observed for the fully extended helical monomer. This was explained in terms of a larger variety of microenvironmental conditions that the tryptophan residue experiences during its excited state. However, the conformation freedom of the peptide can be modulated by varying the micellar size.