The tetrameric protein transthyretin dissociates to a non-native monomer in solution. A novel model for amyloidogenesis.
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In amyloidosis, normally innocuous soluble proteins polymerize to form insoluble fibrils. Amyloid fibril formation and deposition have been associated with a wide range of diseases, including spongiform encephalopathies, Alzheimer's disease, and familial amyloid polyneuropathies (FAP). In certain forms of FAP, the amyloid fibrils are mostly constituted by variants of transthyretin (TTR), a homotetrameric plasma protein implicated in the transport of thyroxine and retinol. The most common amyloidogenic TTR variant is V30M-TTR, and L55P-TTR is the variant associated with the most aggressive form of FAP. Recently, we reported that TTR dissociates to a monomeric species at pH 7.0 and nearly physiological ionic strengths (Quintas, A., Saraiva, M. J., and Brito, R. M. (1997) FEBS Lett. 418, 297-300). Here, we show that the tetramer dissociation is apparently irreversible; and based on intrinsic tryptophan fluorescence and fluorescence quenching experiments, we show that the monomeric species formed upon tetramer dissociation is non-native. We also show, based on 1-anilino-8-naph-thalenesulfonate binding studies, that this monomeric species appears not to behave like a molten globule. These data allowed us to propose a model for TTR amyloidogenesis based on tetramer dissociation occurring naturally under commonly observed physiological solution conditions. (+info)
Eye lens alphaA- and alphaB-crystallin: complex stability versus chaperone-like activity.
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The major lens protein alpha-crystallin is composed of two related types of subunits, alphaA- and alphaB-crystallin, of which the former is essentially lens-restricted, while the latter also occurs in various other tissues. With regard to their respective chaperone capacities, it has been reported that homomultimeric alphaA-crystallin complexes perform better in preventing thermal aggregation of proteins, while alphaB-crystallin complexes protect more efficiently against reduction-induced aggregation of proteins. Here, we demonstrate that this seeming discrepancy is solved when the reduction assay is performed at increasing temperatures: above 50 degrees C alphaA- performs better than alphaB-crystallin also in this assay. This inversion in protective capacity might relate to the greater resistance of alphaA-crystallin to heat denaturation. Infrared spectroscopy, however, revealed that this is not due to a higher thermostability of alphaA-crystallin's secondary structure. Also the accessible hydrophobic surfaces do not account for the chaperoning differences of alphaA- and alphaB-crystallin, since regardless of the experimental temperature alphaB-crystallin displays a higher hydrophobicity. It is argued that the greater complex stability of alphaA-crystallin, as evident upon urea denaturation, and the higher chaperone capacity of alphaB-crystallin at physiological temperatures reflect the evolutionary compromise to obtain an optimal functioning of heteromeric alpha-crystallin as a lens protein. (+info)
Domain separation precedes global unfolding of rhodanese.
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The enzyme rhodanese was investigated for the conformational transition associated with its urea unfolding. When rhodanese was treated with 0 or 3 M urea, the activity was not significantly affected. 4.25 M urea treatment led to a time-dependent loss of activity in 60 min. Rhodanese was completely inactivated within 2 min in 6 M urea. The 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid fluorescence intensity was not significantly increased during 0, 3, and 6 M urea equilibrations, and the fluorescence was dramatically increased with 4.25 M urea, indicating that hydrophobic surfaces are exposed. After 0 and 3 M urea equilibration, rhodanese was not significantly proteolyzed with trypsin. Treatment with 4.25 M urea led to simultaneous formation of major 12-, 15.9-, 17-, and 21.2-kDa fragments, followed by progressive emergence of smaller peptides. The N termini of the 17- and 21.2-kDa bands were those of intact rhodanese. The N terminus of the 15.9-kDa band starts at the end of the interdomain tether. The 12-kDa band begins with either residue 183 or residue 187. The size and sequence information suggest that the 17- and 15.9-kDa bands correspond to the two domains. The 21.2- and 12-kDa bands appear to be generated through one-site tryptic cleavage. It is concluded that urea disrupts interaction between the two domains, increasing the accessibility of the interdomain tether that can be digested by trypsin. The released domains have increased proteolytic susceptibility and produce smaller peptides, which may represent subdomains of rhodanese. (+info)
Thermodynamics of target peptide recognition by calmodulin and a calmodulin analogue: implications for the role of the central linker.
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The thermodynamics of interaction of two model peptides melittin and mastoparan with bovine brain calmodulin (CAM) and a smaller CAM analogue, a calcium binding protein from Entamoeba histolytica (CaBP) in 10 mM MOPS buffer (pH 7.0) was examined using isothermal titration calorimetry (ITC). These data show that CAM binds to both the peptides and the enthalpy of binding is endothermic for melittin and exothermic for mastoparan at 25 degrees C. CaBP binds to the longer peptide melittin, but does not bind to mastoparan, the binding enthalpy being endothermic in nature. Concurrently, we also observe a larger increase in alpha-helicity upon the binding of melittin to CAM when compared to CaBP. The role of hydrophobic interactions in the binding process has also been examined using 8-anilino-1-naphthalene-sulphonic acid (ANS) binding monitored by ITC. These results have been employed to rationalize the energetic consequences of the binding reaction. (+info)
Thermodynamic stability of human lens recombinant alphaA- and alphaB-crystallins.
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Lens alpha-crystallin is a 600-800-kDa heterogeneous oligomer protein consisting of two subunits, alphaA and alphaB. The homogeneous oligomers (alphaA- and alphaB-crystallins) have been prepared by recombinant DNA technology and shown to differ in the following biophysical/biochemical properties: hydrophobicity, chaperone-like activity, subunit exchange rate, and thermal stability. In this study, we studied their thermodynamic stability by unfolding in guanidine hydrochloride. The unfolding was probed by three spectroscopic parameters: absorbance at 235 nm, Trp fluorescence intensity at 320 nm, and far-UV circular dichroism at 223 nm. Global analysis indicated that a three-state model better describes the unfolding behavior than a two-state model, an indication that there are stable intermediates for both alphaA- and alphaB-crystallins. In terms of standard free energy (DeltaG(NU)(H(2)(O))), alphaA-crystallin is slightly more stable than alphaB-crystallin. The significance of the intermediates may be related to the functioning of alpha-crystallins as chaperone-like molecules. (+info)
Differential temperature-dependent chaperone-like activity of alphaA- and alphaB-crystallin homoaggregates.
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alpha-Crystallin, a heteromultimeric protein made up of alphaA- and alphaB-crystallins, functions as a molecular chaperone in preventing the aggregation of proteins. We have shown earlier that structural perturbation of alpha-crystallin can enhance its chaperone-like activity severalfold. The two subunits of alpha-crystallin have extensive sequence homology and individually display chaperone-like activity. We have investigated the chaperone-like activity of alphaA- and alphaB-crystallin homoaggregates against thermal and nonthermal modes of aggregation. We find that, against a nonthermal mode of aggregation, alphaB-crystallin shows significant protective ability even at subphysiological temperatures, at which alphaA-crystallin or heteromultimeric alpha-crystallin exhibit very little chaperone-like activity. Interestingly, differences in the protective ability of these homoaggregates against the thermal aggregation of beta(L)-crystallin is negligible. To investigate this differential behavior, we have monitored the temperature-dependent structural changes in both the proteins using fluorescence and circular dichroism spectroscopy. Intrinsic tryptophan fluorescence quench-ing by acrylamide shows that the tryptophans in alphaB-crystallin are more accessible than the lone tryptophan in alphaA-crystallin even at 25 degrees C. Protein-bound 8-anilinonaphthalene-1-sulfonate fluorescence demonstrates the higher solvent accessibility of hydrophobic surfaces on alphaB-crystallin. Circular dichroism studies show some tertiary structural changes in alphaA-crystallin above 50 degrees C. alphaB-crystallin, on the other hand, shows significant alteration of tertiary structure by 45 degrees C. Our study demonstrates that despite a high degree of sequence homology and their generally accepted structural similarity, alphaB-crystallin is much more sensitive to temperature-dependent structural perturbation than alphaA- or alpha-crystallin and shows differences in its chaperone-like properties. These differences appear to be relevant to temperature-dependent enhancement of chaperone-like activity of alpha-crystallin and indicate different roles for the two proteins both in alpha-crystallin heteroaggregate and as separate proteins under stress conditions. (+info)
Insights into the structure and substrate interactions of the P-glycoprotein multidrug transporter from spectroscopic studies.
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The P-glycoprotein multidrug transporter is a 170-kDa efflux pump which exports a diverse group of natural products, chemotherapeutic drugs, and hydrophobic peptides across the plasma membrane, driven by ATP hydrolysis. The transporter has been proposed to interact with its drug substrates within the membrane environment; however, much remains to be learned about the nature and number of the drug binding site(s). The two nucleotide binding domains are responsible for ATP binding and hydrolysis, which is coupled to drug movement across the membrane. In recent years, P-glycoprotein has been purified and functionally reconstituted in amounts large enough to allow biophysical studies. The use of spectroscopic techniques has led to insights into both its secondary and tertiary structure, and its interaction with nucleotides and drugs. In this review, we will summarise what has been learned by application to purified P-glycoprotein of fluorescence spectroscopy, circular dichroism spectroscopy and infra-red spectroscopy. (+info)
Anion-induced refolding of human serum albumin under low pH conditions.
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We studied the effect of various anions (of acids and salts) on the acid denatured state of HSA by near-UV circular dichroism (CD), far-UV CD, 1-anilinonaphthalene-8-sulfonate (ANS) binding, tryptophan fluorescence and thermal transition. Addition of different acids and salts caused an induction of alpha-helical structure as evident from the increase in the mean residue ellipticity (MRE) value at 222 nm and loss of ANS binding sites exhibited by the decrease in the ANS fluorescence intensity at 480 nm. However, the concentration range of acids/salts required to bring about the transition varied greatly among different acids and salts. Among various acids/salts tested, K(3)Fe(CN)(6) was found to be most effective whereas HCl and KCl were least effective in inducing the properties close to native structure. Further, they followed the electroselectivity series. The near-UV CD spectra showed an increase in MRE towards the native state, whereas the tryptophan fluorescence emission spectra produced a red shift of about 6 nm on addition of KClO(4). The temperature-induced transition in the presence of 40 mM KClO(4) monitored by ellipticity measurements at 222 nm was characterized by the presence of an intermediate state in the temperature range 30-50 degrees C having abundant secondary structure. These results suggest that human serum albumin at low pH and in the presence of acids or salts exists in a partially folded state characterized by native-like secondary structure and tertiary folds. (+info)