Enhanced thermodynamic stability of beta-lactoglobulin at low pH. A possible mechanism.
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ABSTRACT:
SUBMITTER: Kella NK
PROVIDER: S-EPMC1135197 | biostudies-other | 1988 Oct
REPOSITORIES: biostudies-other
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Project description:Ions are involved in multiple biological processes and may exist bound to biomolecules or may be associated with their surface. Although the presence of ions in nucleic acids has traditionally gained more interest, ion-protein interactions, often with a marked dependency on pH, are beginning to gather attention. Here we present a detailed analysis on the binding and distribution of ions around β-lactoglobulin using a constant-pH MD (CpHMD) method, at a pH range 3-8, and compare it with the more traditional Poisson-Boltzmann (PB) model and the existing experimental data. Most analyses used ion concentration maps built around the protein, obtained from either the CpHMD simulations or PB calculations. The requirements of approximate charge neutrality and ionic strength equal to bulk, imposed on the MD box, imply that the absolute value of the ion excess should be half the protein charge, which is in agreement with experimental observation on other proteins ( Proc. Natl. Acad. Sci. U.S.A. 2021, 118, e2015879118) and lends support to this protocol. In addition, the protein total charge (including territorially bound ions) estimated with MD is in excellent agreement with electrophoretic measurements. Overall, the CpHMD simulations show good agreement with the nonlinear form of the PB (NLPB) model but not with its linear form, which involves a theoretical inconsistency in the calculation of the concentration maps. In several analyses, the observed pH-dependent trends for the counterions and co-ions are those generally expected, and the ion concentration maps correctly converge to the bulk ionic strength as one moves away from the protein. Despite the overall similarity, the CpHMD and NLPB approaches show some discrepancies when analyzed in more detail, which may be related to an apparent overestimation of counterion excess and underestimation of co-ion exclusion by the NLPB model, particularly at short distances from the protein.
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Project description:Although bovine beta-lactoglobulin assumes a monomeric native structure at pH 3 in the absence of salt, the addition of salts stabilizes the dimer. Thermodynamics of the monomer-dimer equilibrium dependent on the salt concentration were studied by sedimentation equilibrium. The addition of NaCl, KCl, or guanidine hydrochloride below 1 M stabilized the dimer in a similar manner. On the other hand, NaClO(4) was more effective than other salts by about 20-fold, suggesting that anion binding is responsible for the salt-induced dimer formation, as observed for acid-unfolded proteins. The addition of guanidine hydrochloride at 5 M dissociated the dimer into monomers because of the denaturation of protein structure. In the presence of either NaCl or NaClO(4), the dimerization constant decreased with an increase in temperature, indicating that the enthalpy change (DeltaH(D)) of dimer formation is negative. The heat effect of the dimer formation was directly measured with an isothermal titration calorimeter by titrating the monomeric beta-lactoglobulin at pH 3.0 with NaClO(4). The net heat effects after subtraction of the heat of salt dilution, corresponding to DeltaH(D), were negative, and were consistent with those obtained by the sedimentation equilibrium. From the dependence of dimerization constant on temperature measured by sedimentation equilibrium, we estimated the DeltaH(D) value at 20 degrees C and the heat capacity change (DeltaC(p)) of dimer formation. In both NaCl and NaClO(4), the obtained DeltaC(p) value was negative, indicating the dominant role of burial of the hydrophobic surfaces upon dimer formation. The observed DeltaC(p) values were consistent with the calculated value from the X-ray dimeric structure using a method of accessible surface area. These results indicated that monomer-dimer equilibrium of beta-lactoglobulin at pH 3 is determined by a subtle balance of hydrophobic and electrostatic effects, which are modulated by the addition of salts or by changes in temperature.
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Project description:Bovine beta-lactoglobulin A assumes a dimeric native conformation at neutral pH, while the conformation at pH 2 is monomeric but still native. Beta-lactoglobulin A has a free thiol at Cys121, which is buried between the beta-barrel and the C-terminal major alpha-helix. This thiol group was specifically reacted with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the presence of 1.0 M Gdn-HCI at pH 7.5, producing a modified beta-lactoglobulin (TNB-bIg) containing a mixed disulfide bond with 5-thio-2-nitrobenzoic acid (TNB). The conformation and stability of TNB-bIg were studied by circular dichroism (CD), tryptophan fluorescence, analytical ultracentrifugation, and one-dimensional 1H-NMR. The CD spectra of TNB-bIg indicated disordering of the native secondary structure at pH 7.5, whereas a slight increase in the alpha-helical content was observed at pH 2.0. The tryptophan fluorescence of TNB-bIg was significantly quenched compared with that of the intact protein, probably by the energy transfer to TNB. Sedimentation equilibrium analysis indicated that, at neutral pH, TNB-bIg is monomeric while the intact protein is dimeric. In contrast, at pH 2.0, both the intact beta-lactoglobulin and TNB-bIg were monomeric. The unfolding transition of TNB-bIg induced by Gdn-HCl was cooperative in both pH regions, although the degree of cooperativity was less than that of the intact protein. The 1H-NMR spectrum for TNB-bIg at pH 3.0 was native-like, whereas the spectrum at pH 7.5 was similar to that of the unfolded proteins. These results suggest that modification of the buried thiol group destabilizes the rigid hydrophobic core and the dimer interface, producing a monomeric state that is native-like at pH 2.0 but is molten globule-like at pH 7.5. Upon reducing the mixed disulfide of TNB-bIg with dithiothreitol, the intact beta-lactoglobulin was regenerated. TNB-bIg will become a useful model to analyze the conformation and stability of the intermediate of protein folding.
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Project description:The protein beta-lactoglobulin aggregates into two apparently distinct forms under different conditions: amyloid fibrils at pH values away from the isoelectric point, and spherical aggregates near it. To understand this apparent dichotomy in behavior, we studied the internal structure of the spherical aggregates by employing a range of biophysical approaches. Fourier transform infrared studies show the aggregates have a high beta-sheet content that is distinct from the native beta-lactoglobulin structure. The structures also bind the amyloidophilic dye thioflavin-T, and wide-angle x-ray diffraction showed reflections corresponding to spacings typically observed for amyloid fibrils composed of beta-lactoglobulin. Combined with small-angle x-ray scattering data indicating the presence of one-dimensional linear aggregates at the molecular level, these findings indicate strongly that the aggregates contain amyloid-like substructure. Incubation of beta-lactoglobulin at pH values increasingly removed from the isoelectric point resulted in the increasing appearance of fibrillar species, rather than spherical species shown by electron microscopy. Taken together, these results suggest that amyloid-like beta-sheet structures underlie protein aggregation over a much broader range of conditions than previously believed. Furthermore, the results suggest that there is a continuum of beta-sheet structure of varying regularity underlying the aggregate morphology, from very regular amyloid fibrils at high charge to short stretches of amyloid-like fibrils that associate together randomly to form spherical particles at low net charge.
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Project description:To clarify the pH-dependent conformational transitions of proteins, we propose an approach in which structural changes monitored by heteronuclear sequential quantum correlation (HSQC) spectroscopy were analyzed by using a principal component analysis (PCA). We use bovine beta-lactoglobulin, a protein widely used in protein folding studies, as a target. First, we measured HSQC spectra at various pH values and subjected them to a PCA. The analysis revealed three apparent transitions with pK(a) values of 2.9, 4.9, and 6.8, consistent with previous reports using different methods. Next, Gdn-HCl-induced unfolding was examined by measuring tryptophan fluorescence at various pH values. Between pH 2 and 8, beta-lactoglobulin exhibited a number of structural transitions as well as changes in stability represented by the free energy change of unfolding, DeltaG(U). By combining the NMR and fluorescence results, the change in DeltaG(U) was suggested to result from the decreased pK(a) of some acidic residues. Notably, the native state at neutral pH is destabilized by deprotonation of Glu-89, leading to an increase in the relative population of the intermediate. Thus, the PCA of pH-dependent HSQC spectra provides a more comprehensive understanding of the stability and function of proteins.
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Project description:?-lactoglobulin (LG) contains nine ?-strands (strands A-I) and one ?-helix. Strands A-H form a ?-barrel. At neutral pH, equine LG (ELG) is monomeric, whereas bovine LG (BLG) is dimeric, and the I-strands of its two subunits form an intermolecular ?-sheet. We previously constructed a chimeric ELG in which the sequence of the I-strand was replaced with that of BLG. This chimera did not dimerize. For this study, we constructed the new chimera we call Gyuba (which means cow and horse in Japanese). The amino acid sequence of Gyuba includes the sequences of the BLG secondary structures and those of the ELG loops. The crystal structure of Gyuba is very similar to that of BLG and indicates that Gyuba dimerizes via the intermolecular ?-sheet formed by the two I-strands. Thus, the entire arrangement of the secondary structural elements is important for LG dimer formation.
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