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SDS-induced multi-stage unfolding of a small globular protein through different denatured states revealed by single-molecule fluorescence.

ABSTRACT: Ionic surfactants such as sodium dodecyl sulfate (SDS) unfold proteins in a much more diverse yet effective way than chemical denaturants such as guanidium chloride (GdmCl). But how these unfolding processes compare on a molecular level is poorly understood. Here, we address this question by scrutinising the unfolding pathway of the globular protein S6 in SDS and GdmCl with single-molecule Förster resonance energy transfer (smFRET) spectroscopy. We show that the unfolding mechanism in SDS is strikingly different and convoluted in comparison to denaturation in GdmCl. In contrast to the reversible two-state unfolding behaviour in GdmCl characterised by kinetics on the timescale of seconds, SDS demonstrated not one, but four distinct regimes of interactions with S6, dependent on the surfactant concentration. At ≤1 mM SDS, S6 and surfactant molecules form quasi-micelles on a minute timescale; at millimolar [SDS], the protein denatures through an unfolded/denatured ensemble of highly heterogeneous states on a multi-second timescale; at tens of millimolar of SDS, the protein unfolds into a micelle-packed conformation on the second timescale; and >50 mM SDS, the protein unfolds with millisecond timescale dynamics. We propose a detailed model for multi-stage unfolding of S6 in SDS, which involves at least three different types of denatured states with different level of compactness and dynamics and a continually changing landscape of interactions between protein and surfactant. Our results highlight the great potential of single-molecule fluorescence as a direct probe of nanoscale protein structure and dynamics in chemically complex surfactant environments.

SUBMITTER: Krainer G

PROVIDER: S-EPMC8163379 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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 ?-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 ?-helical structure, which persists in SDS (albeit as isolated helices), enabling aLA to unfold without undergoing major secondary structural changes unlike ?-sheet rich proteins. Nevertheless, the overall unfolding steps are broadly similar to those of the more ?-rich protein ?-lactoglobulin, suggesting that this unfolding model is representative of the general process of SDS-unfolding of proteins.

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SDS-induced multi-stage unfolding of a small globular protein through different denatured states revealed by single-molecule fluorescence.

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