Search

Myoglobin is an -helical globular protein containing two highly conserved tryptophanyl residues at positions 7 and 14 in the N-terminal region. The simultaneous substitution of the two residues impairs the productive folding of the protein making the polypeptide chain highly prone to aggregate forming amyloid fibrils at physiological pH and room temperature. The role played by tryptophanyl residues in driving the productive folding process was investigated by providing structural details at low resolution of compact intermediate of three mutated apomyoglobins, i.e., W7F, W14F and the amyloid forming mutant W7FW14F. In particular, we followed the hydrogen/deuterium exchange rate of protein segments using proteolysis with pepsin followed by mass spectrometry analysis. The results revealed significant differences in the N-terminal region, consisting in an alteration of the physico-chemical properties of the 7-11 segment for W7F and in an increase of local flexibility of the12-29 segment for W14F. In the double trypthophanyl substituted mutant, these effects are additive and impair the formation of native-like contacts and favour inter-chain interactions leading to protein aggregation and amyloid formation at physiological pH.

Importance of the field:The identification of molecules that inhibit protein deposition or reverse fibril formation could open new strategies for therapeutic intervention in misfolding diseases. Numerous compounds have been shown to inhibit amyloid fibril formation in vitro.Among these compounds, tetracycline and the disaccharide trehalose have been reported to inhibit or reverse amyloid aggregation but their efficiency as potential drugs is controversial.Areas covered in this review:The results obtained using tetracycline and trehalose, reported in the last 15 years, are described and discussed.What the reader will gain:The conclusions have important implications for the development of therapeutic agents for protein deposition diseases. If fibrillar proteins contribute to cell degeneration, then the disassembly of fibrils may reverse or slow down disease progression; however, if the action of therapeutic agents produces intermediates of fibrillation and/or products of fibril disaggregation, then their accumulation could be harmful.Take home message:Care should be taken to ensure that strategies aimed at inhibiting fibril formation do not cause a corresponding increase in the concentration of toxic oligomeric species.

Myoglobin is an alpha-helical globular protein that contains two highly conserved tryptophan residues located at positions 7 and 14 in the N-terminal region of the protein. Replacement of both indole residues with phenylalanine residues, i.e. W7F/W14F, results in the expression of an unstable, not correctly folded protein that does not bind the prosthetic group. Here we report data (Congo red and thioflavine T binding assay, birefringence, and electron microscopy) showing that the double Trp/Phe replacements render apomyoglobin molecules highly susceptible to aggregation and amyloid-like fibril formation under physiological conditions in which most of the wild-type protein is in the native state. In refolding experiments, like the wild-type protein, the W7F/W14F apomyoglobin mutant formed a soluble, partially folded helical state between pH 2.0 and pH 4.0. A pH increase from 4.0 to 7.0 restored the native structure only in the case of the wild-type protein and determined aggregation of W7F/W14F. The circular dichroism spectrum recorded immediately after neutralization showed that the polypeptide consists mainly of beta-structures. In conclusion, under physiological pH conditions, some mutations that affect folding may cause protein aggregation and the formation of amyloid-like fibrils.

The tryptophanylemission decay of the mesophilic β-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 β-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. © 2002 John Wiley & Sons, Inc. Biopolymers (Biospectroscopy) 67: 85–95, 2002

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 tryptophanyl emission decay of β‐glycosidase from the extremophilic archaeon Sulfolobus solfataricus(Sβgly) 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 Sβgly has been investigated by observing the changes induced in the two components of the tryptophanyl emission decay. The addition of cyclophellitol to native Sβgly 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 Sβgly allowed us to evaluate some average parameters for each tryptophanyl microenvironment in the Sβgly such as hydrophobicity, structural flexibility, and ability of side chains to act as fluorescence quenchers. These results permitted to divide the tryptophanyl fluorescence of Sβgly 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 β‐galactosidase from Escherichia coli(Cβgal) 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. Proteins 1999;35:163–172. © 1999 Wiley‐Liss, Inc.

The tryptophanyl emission decay of β-glycosidase from the extremophilic archaeon Sulfolobus solfataricus (Sβgly) 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 Sβgly has been investigated by observing the changes induced in the two components of the tryptophanyl emission decay. The addition of cyclophellitol to native Sβgly 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 Sβgly allowed us to evaluate some average parameters for each tryptophanyl microenvironment in the Sβgly such as hydrophobicity, structural flexibility, and ability of side chains to act as fluorescence quenchers. These results permitted to divide the tryptophanyl fluorescence of Sβgly 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 β-galactosidase from Escherichia coli (Cβgal) 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. Proteins 1999;35:163–172. © 1999 Wiley-Liss, Inc.

1.1. The heart ventricle myoglobin of bluefin tuna has been purified to an apparent homogeneity.2.2. The amino acid analysis has revealed only a limited number of substitutions between the myoglobins of yellowfin and bluefin tuna.3.3. The α-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 α-helix content.4.4. 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.5.5. 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.

The conformational dynamics of β-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 β-glycosidase similar to those observed in the absence of detergent. Proteins 27:71–79 © 1997 Wiley-Liss, Inc.

1.1. The skeletal muscle of Bos bubalusL. has been fractionated into three different protein fractions: sarcoplasmic, myofibrillar and insoluble.2.2. The amino acid composition of these fractions has been determined. The content of essential amino acids in the various fractions has been found to be very high in decreasing order from the sarcoplasmic to the myofibrillar and insoluble fraction.3.3. The “chemical score” followed by the indication of the limiting amino acid has been calculated to be 74 (Ileu), 71 (Val) and 65 (Phe), respectively, for the sarcoplasmic, myofibrillar and insoluble fraction.4.4. The high nutritional value of meat fractions from the domestic buffalo from the Mediterranean area is supported by experimental data.

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 × 109M-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 × 109M-1s-1. This value if compared to that determined for the native apoprotein at neutral pH, i.e. 4·0 × 108M-1s-1, indicates that the native-like structure surviving the acid-induced transition possesses a large molecular flexibility.

The conformational dynamics of β‐glycosidase from Sulfolobus solfataricuswas 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 β‐glycosidase similar to those observed in the absence of detergent. Proteins 27:71–79 © 1997 Wiley‐Liss, Inc.

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.

The fluorescence lifetime distribution of 2-p-toluidinyl-6-naphthalene sulfonic acids (TNS) bound to the heme site of apomyoglobin has been examined. The results were compared to those observed for the free fluorophore in isotropic nonviscous solvent. Two different excitation wavelengths were used, i.e. 290 and 350 nm. The results showed that the distribution of TNS bound to apomyoglobin is wider than that of the free fluorophore, thus indicating the existence of a large number of conformational substates originating from the interaction between TNS and the protein matrix. The comparison of the distribution obtained at two different excitation wavelengths allowed the emission arising from conformational substates, in which the excited state of fluorophore moiety has a higher probability to be populated by Forster energy transfer mechanism, to be distinguished.