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Principal component analysis of the pH-dependent conformational transitions of bovine beta-lactoglobulin monitored by heteronuclear NMR.

ABSTRACT: 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.

SUBMITTER: Sakurai K

PROVIDER: S-EPMC2000507 | biostudies-literature | 2007 Sep

REPOSITORIES: biostudies-literature

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Principal component analysis of the pH-dependent conformational transitions of bovine beta-lactoglobulin monitored by heteronuclear NMR.

Sakurai Kazumasa K Goto Yuji Y

Proceedings of the National Academy of Sciences of the United States of America 20070918 39

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) val ...[more]

PMID: 17878316

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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.

Dataset Information

Principal component analysis of the pH-dependent conformational transitions of bovine beta-lactoglobulin monitored by heteronuclear NMR.

Publications

Principal component analysis of the pH-dependent conformational transitions of bovine beta-lactoglobulin monitored by heteronuclear NMR.

Similar Datasets

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