Multi-Step Unfolding and Rearrangement of α-Lactalbumin by SDS Revealed by Stopped-Flow SAXS

Abstract

Interactions between proteins and surfactants are both of fundamental interest and relevant for applications in food, cosmetics and detergency. The anionic surfactant sodium dodecyl sulfate (SDS) denatures essentially all proteins. Denaturation typically involves a number of distinct steps where growing numbers of SDS molecules bind to the protein, as seen in multidisciplinary approaches combining several complementary techniques. We adopt this approach to study the SDS-induced unfolding of Ca²⁺-depleted α-lactalbumin (aLA), a protein particularly sensitive toward denaturation by surfactants. By combining stopped-flow mixing of protein and surfactant solutions with stopped-flow synchrotron small-angle X-ray scattering (SAXS), circular dichroism (CD) and Trp fluorescence, together with information from previous calorimetric studies, we construct a detailed picture of the unfolding process at the level of both protein and surfactant. A protein-surfactant complex is formed within the dead time of mixing (2.5 ms). Initially a cluster of SDS molecules binds asymmetrically, i.e., to one side of the protein, after which aLA redistributes around the SDS cluster. This occurs in two kinetic steps where the complex grows in number of both SDS and protein molecules, concomitant with protein unfolding. During these steps, the core-shell complex undergoes changes in shell thickness as well as core shape and radius. The entire process is very sensitive to SDS concentration and completes within 10 s at an SDS:aLA ratio of 9, decreasing to 0.2 s at 60 SDS:aLA. The number of aLA molecules per SDS complex drops from 1.9 to 1.0 over this range of ratios. While both CD and Trp kinetics reveal a fast and a slow conformational transition, only the slow transition is observed by SAXS, indicating that the protein-SDS complex (which is monitored by SAXS) adjusts to the presence of the unfolded protein. We attribute the rapid unfolding of aLA to its predominantly α-helical structure, which persists in SDS (albeit as isolated helices), enabling aLA to unfold without undergoing major secondary structural changes unlike β-sheet rich proteins. Nevertheless, the overall unfolding steps are broadly similar to those of the more β-rich protein β-lactoglobulin, suggesting that this unfolding model is representative of the general process of SDS-unfolding of proteins.

Keywords: circular dichroism; fluorescence; protein-SDS interactions; stopped-flow kinetics; supramolecular complex structures; synchrotron SAXS; unfolding mechanisms; α-lactalbumin.

Figure 1

Illustration of the structural model applied to fit the SAXS data (see text).

Figure 2

(A) Trp fluorescence vs. wavelength for SDS:aLA mixing ratios of 0, 9, 27, 40, and 60 (black, red, green, blue, and purple points). (B) Far-UV CD spectra for SDS:aLA mixing ratios of 0, 9, 27, 40, and 60 after equilibration. (C) Near-UV CD spectra for SDS:aLA mixing ratios of 0, 9, 27, 40, and 60 after equilibration. (D,E) Trp (D) and near-UV CD (E) time profiles for 1:1 stopped-flow mixing of aLA and SDS at 9–60 SDS:aLA mixing ratios. The lines are fits to double exponential functions as described in the text.

Figure 3

Half times t₁ and t₂ for aLA unfolding and formation of surfactant-protein complexes, observed by Trp fluorescence, near-UV CD, and SAXS. Filled and empty symbols of the same kind (e.g., square) represent slow and fast phases associated with that technique (for squares this is Trp fluorescence). The SAXS half times are obtained from the time evolution of parameters describing the number of aLA molecules per complex, N_aLA, and the offset of the protein shell around the micelle core, s. Note that the stoichiometries refer to the compositions of the SDS:aLA complexes [based on ITC values Otzen et al., 2009] rather than bulk ratios. For the smallest SDS_total:aLA ratio of 9 (which leads to a ratio of 6 SDS:aLA in the protein complex), CD (brown empty circle) and N_aLA values (cyan empty triangle) showed an additional fast exponential decay.

Figure 4

SAXS data for solutions of 28 mM SDS (orange squares), 0.7 mM aLA (blue circles) and a 1:1 mixture of the two (pink triangles), resulting in an SDS:aLA mixing ratio of 40.

Figure 5

(A–D) Stopped-flow SAXS data plotted as scattering intensities vs. the momentum transfer q, collected after mixing SDS and aLA solution to obtain SDS:aLA ratios of 9, 27, 40, and 60, respectively. Time increases from blue to yellow. Selected data sets are shown. Corresponding staggered plots are shown in the SI (Figure S1) for better comparison of data and fits.

Figure 6

Parameters from model fits to kinetic SAXS data after stopped-flow mixing of aLA and SDS in mixing ratios SDS:aLA 9 (red points), 27 (green points), 40 (blue points), and 60 (purple points). (A) The shell thickness, D. (B) The core radius R_core. (C) Axis ratio of ellipsoidal core, ε. (D) Shift s of the core relative to the center of the outer ellipsoid in the core-shell model (see text). (E) Number of SDS molecules per micelle in protein-surfactant complex, N_SDS. (F) Number of aLA molecules per complex, N_αLA.

Figure 7

Schematic representation of the unfolding process after mixing of aLA (red) and SDS (blue).