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.

Scheme. 1.

Experimental design.

Fig. 1.

pH titration monitored by HSQC spectrum. (A) A close-up view of superposition of HSQC spectra acquired at pH 2.4–8.1. The red, orange, green, and blue spectra were acquired at pH 2.4, 5.0, 6.7, and 8.1, respectively. Signal displacements of residues are indicated by arrows. See SI Fig. 8 for all regions. (B) An example of signal displacements of Gln-5. The error bars for δ_H and δ_N are set to be 0.02 and 0.1 ppm, respectively, which indicate the standard linewidth of the signals. The solid lines are reconstructed traces from the results of the PCA. The letters are marked at the estimated signal positions for each state. (C and D) A Hue Plot for all of the residues (C) and the scale figure (D). The bars and circles in C indicate the positions of β-strands, the α-helix, and residues involved in the dimer interface, respectively.

Fig. 2.

Fitting of PCs. The pH dependencies of the first, second, third, and fourth PCs are shown. The lines are theoretical curves based on Eq. 2, in which only the first, second, and third PCs were considered.

Fig. 3.

The result of the PCA. (A–C) CSDs for the transitions at pH 2.9 (A), 4.9 (B), and 6.8 (C). Heights and colorations of the bars indicate the distances and the directions of the signal displacements, respectively. The colorations obeying the Hue scale were indicated in Fig. 1C. (D–F) Mapping of the obtained CSDs at each pH on the crystal structure.

Fig. 4.

pH- and Gdn-HCl-dependent fluorescence spectra of βLG. (A and B) Fluorescence spectra of wild-type βLG in the presence of various concentrations of Gdn-HCl at pH 3.0 (A) and 7.0 (B). The concentration of Gdn-HCl increases from 0 M (red) to 6.0 M (purple) in steps of 0.24 M as guided by arrows. (C) The result of the global fitting. The markers indicate the fluorescence intensities at 330 nm at pH 2.0 (red), 4.0 (green), 6.0 (blue), and 8.0 (purple), respectively, and the lines indicate the theoretical curves. (D) The ΔG profiles for the N (red) and I (blue) states. The solid and broken lines are of the wild-type and A34C βLG, respectively.

Fig. 5.

PFs at pD_r 3.0 (red), 4.7 (green), and 6.9 (blue). The broken lines indicate the maximum values of PF obtained at each pD_r. The residues with the maximum PF values at any pH are indicated by the gray background. The lower triangles indicate the residues that did not show observable H/D exchange at pD_r 4.9 during the measurement (6 months).