Getting Smaller by Denaturation: Acid-Induced Compaction of Antibodies

Abstract

Protein denaturation is a ubiquitous process that occurs both in vitro and in vivo. While our molecular understanding of the denatured structures of proteins is limited, it is commonly accepted that the loss of unique intramolecular contacts makes proteins larger. Herein, we report compaction of the immunoglobulin G1 (IgG1) protein upon acid denaturation. Small-angle X-ray scattering coupled with size exclusion chromatography revealed that IgG1 radii of gyration at pH 2 were ∼75% of those at a neutral pH. Scattering profiles showed a compact globular shape, supported by analytical ultracentrifugation. The acid denaturation of proteins with a decrease in size is energetically costly, and acid-induced compaction requires an attractive force for domain reorientation. Such intramolecular aggregation may be widespread in immunoglobulin proteins as noncanonical structures. Herein, we discuss the potential biological significance of these noncanonical structures of antibodies.

Figure 1

MAb-A pH dependence of small-angle X-ray scattering (SAXS) at 25 °C. Concentration-normalized SAXS profiles are presented as (a) log–log and (b) Kratky plots. The protein concentration was 3.5 ± 0.1 mg/mL. The data are vertically shifted for the sake of clarity. The black line shows the theoretical SAXS profile of the IgG1 model (Protein Data Bank entry 1HZH). The arrows in panel a indicate the shoulders at q values of ∼0.08 and ∼0.16 Å^–1.

Figure 2

Size exclusion chromatography small-angle X-ray scattering (SEC-SAXS) analysis of mAb-A at pH 2. The data for mAb-A at 0 and 0.2 M NaCl are colored gray and cyan, respectively. (a) Overlaid elution SAXS and UV profiles where the SAXS-derived chromatogram is an integrated SAXS intensity vs retention time. The elution UV profiles were acquired by simultaneous recording of the mAb-A absorbance at 280 nm. (b) SAXS profiles of monomeric mAb-A. The scattering for mAb-A in 0.2 M NaCl was multiplied by 10² for the sake of clarity. Theoretical scatterings of the triaxial ellipsoid (red solid line) and ellipsoid (dashed black line) with best fit parameters for mAb-A are shown. An elliptical object indicates where a, b, and c are the length parameters. (c) Dimensionless Kratky plots of IgG1 SAXS profiles and theoretical scattering. (d) Distance-distribution function, P(r), determined by direct Fourier transformation of the SAXS profiles. P(r) values of the triaxial ellipsoid (red solid line) and ellipsoid (black dashed line) were calculated from the scatterings in panel b. The P(r) of native mAb-A at pH 7.1 (Figure S1e) is overlaid for comparison. This P(r) was scaled such that the area is equivalent to the P(r) of the mAb-A at pH 2 and 0 M NaCl. (e) Point cloud representation of the triaxial ellipsoids using best fit parameters for the scatterings of mAb-A at 0 and 0.2 M NaCl. A canonical IgG1 structure, in which the C_α atoms (red point clouds) are placed to fit with the triaxial ellipsoids with characteristic lengths. (f) 3D reconstructed models of mAb-A at pH 2 using ab initio electron density determination (DENSS). The canonical mAb-A models are also shown, indicating the ab initio model reconstructed from the SAXS data (light pink), overlaid with the atomic coordinates of IgG1 (red ribbons). The 3D model files are available in the Supporting Information. All of the volumes inside the model surfaces are set to 258 000 ų, where the Porod volume was determined for the scattering of mAb-A at 0 M NaCl. (g) Dependence of the mAb-A R_g at pH 2 on incubation time and NaCl concentration. The R_g of native mAb-A at pH 7 (with 0 M NaCl and 0.1 M NaCl) is indicated for comparison. The scattering data are deposited in the Small Angle Scattering Biological Data Bank (SASBDB) as entries SASDQ99 (https://www.sasbdb.org/data/SASDQ99) and SASDQA9 (https://www.sasbdb.org/data/SASDQA9).

Figure 3

Dependence of R_g on protein chain length. The data in the literature include those for native (filled green circles), native oligomeric (filled dark orange circles), and denatured (black triangles, where filled and empty triangles indicate disulfide-free and disulfide-bonded forms, respectively) proteins, curated by Po-Min Shih et al. The dotted lines represent the power law scaling of R_g = R₀L^ξ, where R₀ is a prefactor (2.04, native; 4.47, native oligomer; and 2.05, denatured), L is the amino acid (a.a.) chain length, and ξ is the scaling exponent (0.45, native; 0.32, native oligomer; and 0.58, denatured). The R_g of mAb-A at pH 7 (empty red circle), the R_g of mAb-A at pH 2 with 0.2 M NaCl (empty red triangle), and the R_g of the mAb-A dimer at pH 2 with 0.2 M NaCl (empty red diamond) determined in this study are overlaid onto previously reported results. The R_g value of mAb-A at pH 7 is given at an infinite dilution of the protein (Figure S1h).

Figure 4

Effects of acid on IgG structure. (a) Charge distribution in IgG1 at various pHs. The electrostatic properties were calculated using the pK_a predictor PROPKA^, and the adaptive Poisson–Boltzmann solver (APBS) software using the atomic coordinates for IgG1 and the mAb-A sequence. (b) Conformational states of the IgG domains at pH 2.0–2.6 (with or without salts) in the context of the isolated domain, Fc/Fab regions, and full-length IgG reported in the literature. The following references are presented in the table: ^apH 2.0,^baglycosylated C_H2 at pH 2,^cpH 2,^daglycosylated Fc at pH 2.6,^eaglycosylated Fc at pH 2.5,^fglycosylated Fc at pH 2.6,^gpH 2,^hpH 2.0,ⁱpH 2,^jpH 2.5,^kpH 2.1,^lglycosylated Fc at pH 2.0,^mpH 2.0, and ⁿpH 2.0. The canonical IgG1 structure, the domains thereof (C_H3, C_H2, C_H1, C_L, V_H, and V_L), and the regions (Fc and Fab) are presented.