Dataset Information

Unfolding bovine α-lactalbumin with T-jump: Characterizing disordered intermediates via time-resolved x-ray solution scattering and molecular dynamics simulations.

ABSTRACT: The protein folding process often proceeds through partially folded transient states. Therefore, a structural understanding of these disordered states is crucial for developing mechanistic models of the folding process. Characterization of unfolded states remains challenging due to their disordered nature, and incorporating multiple methods is necessary. Combining the time-resolved x-ray solution scattering (TRXSS) signal with molecular dynamics (MD), we are able to characterize transient partially folded states of bovine α-lactalbumin, a model system widely used for investigation of molten globule states, during its unfolding triggered by a temperature jump. We track the unfolding process between 20 µs and 70 ms and demonstrate that it passes through three distinct kinetic states. The scattering signals associated with these transient species are then analyzed with TRXSS constrained MD simulations to produce protein structures that are compatible with the input signals. Without utilizing any experimentally extracted kinetic information, the constrained MD simulation successfully drove the protein to an intermediate molten globule state; signals for two later disordered states are refined to terminal unfolded states. From our examination of the structural characteristics of these disordered states, we discuss the implications disordered states have on the folding process, especially on the folding pathway. Finally, we discuss the potential applications and limitations of this method.

SUBMITTER: Hsu DJ

PROVIDER: S-EPMC7943248 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Unfolding bovine α-lactalbumin with T-jump: Characterizing disordered intermediates via time-resolved x-ray solution scattering and molecular dynamics simulations.

Hsu Darren J DJ Leshchev Denis D Kosheleva Irina I Kohlstedt Kevin L KL Chen Lin X LX

The Journal of chemical physics 20210301 10

The protein folding process often proceeds through partially folded transient states. Therefore, a structural understanding of these disordered states is crucial for developing mechanistic models of the folding process. Characterization of unfolded states remains challenging due to their disordered nature, and incorporating multiple methods is necessary. Combining the time-resolved x-ray solution scattering (TRXSS) signal with molecular dynamics (MD), we are able to characterize transient partia ...[more]

PMID: 33722011

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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 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.

Dataset Information

Unfolding bovine α-lactalbumin with T-jump: Characterizing disordered intermediates via time-resolved x-ray solution scattering and molecular dynamics simulations.

Publications

Unfolding bovine α-lactalbumin with T-jump: Characterizing disordered intermediates via time-resolved x-ray solution scattering and molecular dynamics simulations.

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