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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.

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.

The perturbation induced by mono- and divalent cations on the thermophilicity and thermostability of Solfolobus solfataricus beta-glycosidase, a hyperthermophilic tetrameric enzyme, has been investigated by spectroscopic and computational simulation methods to ascertain the Hofmeister effects on two strategic protein regions identified previously. Specifically, (1) an extra segment (83-124), present only in the sequence of hyperthermophilic glycosidases and recognized as an important thermostability determinant for the enzyme structure; and (2) a restricted area of the subunit interface responsible for the quaternary structure maintenance. Mono- and divalent cations inhibit to a different extent the beta-glycosidase activity, whose kinetic constants show an apparent competitive inhibition of the catalytic process that reflects the Hofmeister order. The thermostability is also affected by the nature and charge of the cations, reaching maximal effects for the case of Mg(2+). Fourier transform infrared spectroscopy has revealed very small changes in the protein secondary structure in the presence of the investigated cations at 20 degrees C, while large effects on the protein melting temperatures are observed. Computational analysis of the enzyme structure has identified negative patches on the accessible surface of the two identified regions. Following the Hofmeister series, cations weaken the existing electrostatic network that links the extra segment to the remaining protein matrix. In particular, the perturbing action of cations could involve the ionic pair interactions E107-R245 and E109-R185, thus leading to a local destructuring of the extra segment as a possible starting event for thermal destabilization. A detailed investigation of the electrostatic network at the A-C intermolecular interface of Sbetagly after energy minimization suggests that cations could cause a strong attenuation of the ion pair interactions E474-K72 and D473-R402, with consequent partial dissociation of the tetrameric structure.

Multiple sequence alignment on 73 proteins belonging to glycosyl hydrolase family 1 reveals the occurrence of a segment (83-124) in the enzyme sequences from hyperthermophilic archaea bacteria, which is absent in all the mesophilic members of the family. The alignment of the known three-dimensional structures of hyperthermophilic glycosidases with the known ones from mesophilic organisms shows a similar spatial organizations of beta-glycosidases except for this sequence segment whose structure is located on the external surface of each of four identical subunits, where it overlaps two alpha-helices. Site-directed mutagenesis substituting N97 or S101 with a cysteine residue in the sequence of beta-glycosidase from hyperthermophilic archaeon Sulfolobus solfataricus caused some changes in the structural and dynamic properties as observed by circular dichroism in far- and near-UV light, as well as by frequency domain fluorometry, with a simultaneous loss of thermostability. The results led us to hypothesize an important role of the sequence segment present only in hyperthermophilic beta-glycosidases, in the thermal adaptation of archaea beta-glycosidases. The thermostabilization mechanism could occur as a consequence of numerous favorable ionic interactions of the 83-124 sequence with the other part of protein matrix that becomes more rigid and less accessible to the insult of thermal-activated solvent molecules.

This paper reports a very simple and fast method to collect eluates with high amounts of hydroxytyrosol, biotransforming Olea europaea L. leaf extract by a thermophilic beta-glycosidase immobilized on chitosan. Some phenolic compounds in the leaf tissue and in the eluates obtained by biotransformation are identified. To propose the eluates as natural substances from a vegetal source, their antioxidant properties have been compared with those of the leaf extract from which they are originated. The eluates possess a higher concentration of simple phenols, characterized by a stronger antioxidant capacity, than those available in extra virgin olive oils and in many tablets of olive leaf extracts, commercially found as dietetic products and food integrators.

The local and global dynamics of the Sulfolobus solfataricus beta-glycosidase were studied by electron spin resonance and time-resolved fluorescence techniques. For electron paramagnetic resonance (EPR) investigations, the protein was covalently modified by the maleimido nitroxide spin label, which is specific for cysteine -SH groups, at position 344 and at position 101, where Ser-101 was changed into a cysteine by site-directed mutagenesis. The greater reactivity of exposed Cys-101 suggested the exclusive modification of this amino acid compared with Cys-344. The labeled proteins underwent temperature perturbation in the range 290-335 K and the values of the spin-label rotation correlation frequencies (nu(c)) ranged from 6 x 10(7) to 2 x 10(8) sec(-1) for the protein labeled at position C344 and from 5.62 x 10(7) to 1.10 x 10(8) sec(-1) for the protein labeled at C101. These rotation correlation values are related to the local dynamic characteristics of the protein matrix. The temperature dependence of rotation correlation frequencies expressed in terms of Arrhenius coordinates (log (nu(c)) vs. 1/T) for the protein labeled at C344 exhibited a linear dependence but with a change in the slope at 311 K. For the protein labeled at C101, no change in the slope was observed at the same temperature. General dynamic information was deduced from the analysis of the fluorescence emission decay of the tryptophanyl residues that are present in each region of the protein structure. Fluorescence data analysis highlighted a bimodal distribution of fluorescence lifetimes arising from the contribution of two emitting groups: one consisting of closely clustered tryptophans responsible for the long-lived emission component (7.1 nsec) and the other composed of tryptophans nearer to the protein surface, which can be associated to the short-lived component (2.5 nsec). The temperature dependence of lifetime distribution parameters linked to the long-lived and short-lived components, expressed in Arrhenius coordinates, showed two different points in which the change in the slope occurred (i.e., 328 K and 338 K, respectively). The Arrhenius analysis of data provided the activation energy relative to the conformational changes characterizing the local and global movements running through the protein matrix.

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.

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 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 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.

Interferon alpha (IFNalpha) induces both apoptosis and a counteracting epidermal growth factor Erk-dependent survival response in cancer cells. In this report, IFNalpha increased eukaryotic elongation factor 1A (eEF-1A) protein expression by inhibition of eEF-1A degradation via a proteasome-dependent pathway. The reduction of the expression level of eEF-1A by RNA interference enhanced the apoptosis induced by IFNalpha on the same cells. Moreover, IFNalpha induced the phosphorylation of both serine and threonine in eEF-1A. These effects were paralleled by an increased co-immunoprecipitation and colocalization of eEF-1A with C-Raf. The suppression of C-Raf kinase activity with the inhibitor BAY 43-9006 completely antagonized the increase of both eEF-1A phosphorylation and expression and of C-Raf/eEF-1A colocalization induced by IFNalpha and enhanced apoptosis and eEF-1A ubiquitination. Cell transfection with the mutated K48R ubiquitin increased EF-1A expression and desensitized tumor cells to the modulating effects of IFNalpha. The dynamic simulation of 3Dstructure of eEF-1A identified putative serine and threonine phosphorylation sites. In conclusion, the interaction between eEF-1A and C-Raf increases eEF-1A stability and induces a survival activity.

The exposure to non-thermal microwave electromagnetic field (MW-EMF) at 1.95 MHz, a frequency used in mobile communication, affects the refolding kinetics of eukaryotic proteins (Mancinelli et al., 2004). On these basis we have evaluated the in vivo effect of MW-EMF in human epidermoid cancer KB cells. We have found that MW-EMF induces time-dependent apoptosis (45% after 3 h) that is paralleled by an about 2.5-fold decrease of the expression of ras and Raf-1 and of the activity of ras and Erk-1/2. Although also the expression of Akt was reduced its activity was unchanged likely as a consequence of the increased expression of its upstream activator PI3K. In the same experimental conditions an about 2.5-fold increase of the ubiquitination of ras and Raf-1 was also found and the addition for 12 h of proteasome inhibitor lactacystin at 10 microM caused an accumulation of the ubiquitinated isoforms of ras and Raf-1 and counteracted the effects of MW-EMF on ras and Raf-1 expression suggesting an increased proteasome-dependent degradation induced by MW-EMF. The exposure of KB cells to MW-EMF induced a differential activation of stress-dependent pathway with an increase of JNK-1 activity and HSP70 and 27 expression and with a reduction of p38 kinase activity and HSP90 expression. The overexpression of HSP90 induced by transfection of KB cells with a plasmid encoding for the factor completely antagonized the apoptosis and the inactivation of the ras --> Erk-dependent survival signal induced by MW-EMF. Conversely, the inhibition of Erk activity induced by 12 h exposure to 10 mM Mek-1 inhibitor U0126 antagonized the effects induced by HSP90 transfection on apoptosis caused by MW-EMF. In conclusion, these results demonstrate for the first time that MW-EMF induces apoptosis through the inactivation of the ras --> Erk survival signaling due to enhanced degradation of ras and Raf-1 determined by decreased expression of HSP90 and the consequent increase of proteasome dependent degradation.

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.

The fluorescence emission decays of single-tryptophan-containing peptides of different chain lengths in their unfolded state were investigated in the frequency domain. The data were analyzed using different functions, i.e., exponential fit and probability-density functions of different shape. We found that unimodal Lorentzian distributions best describe the fluorescence decays. This finding agrees with the point of view, now broadly accepted, that rapid motions exist in polypeptides. As a consequence of this flexibility, a large variety of conformations, with an unequal perturbation of tryptophan in its excited state, is generated. The lifetime distribution center was independent of the length of the polypeptide chain but strongly related to the nature of the amino acid residues located in the proximity of the tryptophan in the primary structure. The full width at half maximum, W, of the lifetime distribution was found to be related to the length of unfolded polypeptide by the empirical logarithmic relationship W = 0.83 log n, where n indicates the number of residues. For short peptides, a single lifetime or a narrow range of lifetimes is observed because of the fast relaxation of the tryptophanyl environment. On peptide lengthening, the spectrum of conformations, which the peptide can assume, increases; this causes a complex fluorescence decay represented by a lifetime distribution. For long polypeptide chains, the motions of the regions far from tryptophan do not significantly perturb the chromophore environment.

Bax, a multi-domain protein belonging to the large family of Bcl-2 proteins, has a pivotal role for the initiation of the cytochrome c-mediated apoptosis, a vital physiologic process to eliminate damaged or unwanted cells. In response to specific stimuli Bax translocates from cytosol to mitochondria outer membrane where a process of oligomerization occurs with pore formation through which cytochrome c and other death molecules escape. The pro-death action of Bax is regulated by the interaction with other pro-survival proteins. However, the conformational changes and the structural details necessary for homo and hetero interaction with other regulating proteins are largely unknown. This article reports a combined investigation of molecular dynamics (MD) simulation and automated docking that evidence the molecular regions of Bax involved in the binding with anti-apoptotic exapeptide (Bip) designed from Ku70, a subunit of the protein complex essential for non-homologous DNA repair but that inhibits also the Bax translocation to mitochondria. Since Bip suppresses apoptosis induced by several anti-cancer drugs, it appears relevant to achieve a better understanding of the structural and dynamical aspects that characterize the Bip–Bax complex in view of potential therapeutic implications. The present results show that the Bax region with the highest affinity for Bip is located in proximity of BH3 homology domain of Bax and also involves the α-helices 1 and 8. Moreover, the comparison of essential motions of Bax at 300 and 400 K before and after the formation of the complex with Bip evidences how the binding with the exa-peptide affects the collective motions of specific molecular districts of Bax considered to have functional relevance. J. Cell. Biochem. © 2006 Wiley-Liss, Inc.

Non-thermal effects induced by exposure to microwave electromagnetic field (MW-EMF) at 1.95 MHz, a frequency used in mobile communication, have been observed on the refolding kinetics of the heme binding site in an intracellular protein: tuna myoglobin, starting from acidic conditions. We have selected myoglobin because it can be considered a good model to study protein interactions with MW-EMF for its well-known high-resolution crystallographic structure. Myoglobin solutions at pH 3.0 were subjected to 3 h exposure to microwave field (with a specific absorption rate of 51 +/- 1 mW/g); the heme site refolding has been followed by measuring the molecular absorption in the Soret spectral region and the data were fitted to a bi-exponential model. The kinetics of exposed samples appear to be slowered by MW-EMF action. Moreover, the tryptophanyl lifetime distribution of the exposed protein, as deduced by the analysis of the fluorescence emission decay from its single tryptophan, appears sharper if compared to non-exposed protein samples. This observation suggests that the presence of MW-EMF could affect the propensity of protein molecules to populate specific conformational substates among which myoglobin molecules fluctuate at acidic pH. Changes in the structural fluctuation caused by MW perturbation can affect differently the aggregation process that occurs competitively during the protein folding, so representing a potential risk for protein "misfolding." These data suggest that MW-EMF could have also biochemical and, consequently, biological effects on eukaryotic cells that are still under investigation.

The global uptake of mobile communication emphasizes the question about possible adverse consequences of the exposure to low-level radiofrequency radiation from mobile phones on human health as result of so-called "non-thermal effects". In order to state safety guidelines it seems appropriate to start by excluding, if possible, non-specific effects on structural and dynamic properties of fundamental biomolecules such as proteins. Proteins are flexible polyelectrolytes; thus, they are susceptible, in principle, to the action of electromagnetic fields. In this article, we investigated the effects of microwaves on structural and functional properties of Tunnus tynnus myoglobin at 1.95 GHz, a frequency used by new wireless microwave communication systems. The protein solution was exposed for 2.5 h to 51 mW/g SAR (specific absorption rate) level. Measurements of absorption spectroscopy, circular dichroism and fluorescence emission decay in the frequency domain do not exhibit any influence of the radiation on the native structural state of protein macromolecules.

The tryptophanyl emission decay of the mesophilic beta-galactosidase from Aspergillus oryzae free in buffer and entrapped in agarose gel is investigated as a function of temperature and compared to that of the hyperthermophilic enzyme from Sulfolobus solfataricus. Both enzymes are tetrameric proteins with a large number of tryptophanyl residues, so the fluorescence emission can provide information on the conformational dynamics of the overall protein structure rather than that of the local environment. The tryptophanyl emission decays are best fitted by bimodal Lorentzian distributions. The long-lived component is ascribed to close, deeply buried tryptophanyl residues with reduced mobility; the short-lived one arises from tryptophanyl residues located in more flexible external regions of each subunit, some of which are involved in forming the catalytic site. The center of both lifetime distribution components at each temperature increases when going from the free in solution mesophilic enzyme to the gel-entrapped and hyperthermophilic enzyme, thus indicating that confinement of the mesophilic enzyme in the agarose gel limits the freedom of the polypeptide chain. A more complex dependence is observed for the distribution widths. Computer modeling techniques are used to recognize that the catalytic sites are similar for the mesophilic and hyperthermophilic beta-galactosidases. The effect due to gel entrapment is considered in dynamic simulations by imposing harmonic restraints to solvent-exposed atoms of the protein with the exclusion of those around the active site. The temperature dependence of the tryptophanyl fluorescence emission decay and the dynamic simulation confirm that more rigid structures, as in the case of the immobilized and/or hyperthermophilic enzyme, require higher temperatures to achieve the requisite conformational dynamics for an effective catalytic action and strongly suggest a link between conformational rigidity and enhanced thermal stability.

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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.