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Unfolding of beta-sheet proteins in SDS.

ABSTRACT: Beta-sheet proteins are particularly resistant to denaturation by sodium dodecyl sulfate (SDS). Here we compare unfolding of two beta-sandwich proteins TNfn3 and TII27 in SDS. The two proteins show different surface electrostatic potential. Correspondingly, TII27 unfolds below the critical micelle concentration via the formation of hemimicelles on the protein surface, whereas TNfn3 only unfolds around the critical micelle concentration. Isothermal titration calorimetry confirms that unfolding of TII27 sets in at lower SDS concentrations, although the total number of bound SDS molecules is similar at the end of unfolding. In mixed micelles with the nonionic detergent dodecyl maltoside, where the concentration of monomeric SDS is insignificant, the behavior of the two proteins converges. TII27 unfolds more slowly than TNfn3 in SDS and follows a two-mode behavior. Additionally TNfn3 shows inhibition of SDS unfolding at intermediate SDS concentrations. Mutagenic analysis suggests that the overall unfolding mechanism is similar to that observed in denaturant for both proteins. Our data confirm the kinetic robustness of beta-sheet proteins toward SDS. We suggest this is related to the inability of SDS to induce significant amounts of alpha-helix structure in these proteins as part of the denaturation process, forcing the protein to denature by global rather than local unfolding.

SUBMITTER: Nielsen MM

PROVIDER: S-EPMC1853130 | biostudies-literature | 2007 May

REPOSITORIES: biostudies-literature

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Unfolding of beta-sheet proteins in SDS.

Nielsen Mette M MM Andersen Kell K KK Westh Peter P Otzen Daniel E DE

Biophysical journal 20070309 10

Beta-sheet proteins are particularly resistant to denaturation by sodium dodecyl sulfate (SDS). Here we compare unfolding of two beta-sandwich proteins TNfn3 and TII27 in SDS. The two proteins show different surface electrostatic potential. Correspondingly, TII27 unfolds below the critical micelle concentration via the formation of hemimicelles on the protein surface, whereas TNfn3 only unfolds around the critical micelle concentration. Isothermal titration calorimetry confirms that unfolding of ...[more]

PMID: 17351005

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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.

Project description:The fact that natural beta-sheet proteins are usually soluble but that fragments or designs of beta structure usually aggregate suggests that natural beta proteins must somehow be designed to avoid this problem. Regular beta-sheet edges are dangerous, because they are already in the right conformation to interact with any other beta strand they encounter. We surveyed edge strands in a large sample of all-beta proteins to tabulate features that could protect against further beta-sheet interactions. beta-barrels, of course, avoid edges altogether by continuous H-bonding around the barrel cylinder. Parallel beta-helix proteins protect their beta-sheet ends by covering them with loops of other structure. beta-propeller and single-sheet proteins use a combination of beta-bulges, prolines, strategically placed charges, very short edge strands, and loop coverage. beta-sandwich proteins favor placing an inward-pointing charged side chain on one of the edge strands where it would be buried by dimerization; they also use bulges, prolines, and other mechanisms. One recent beta-hairpin design has a constrained twist too great for accommodation into a larger beta-sheet, whereas some beta-sheet edges are protected by the bend and reverse twist produced by an Lbeta glycine. All free edge strands were seen to be protected, usually by several redundant mechanisms. In contrast, edge strands that natively form beta H-bonded dimers or rings have long, regular stretches without such protection. These results are relevant to understanding how proteins may assemble into beta-sheet amyloid fibers, and they are especially applicable to the de novo design of beta structure. Many edge-protection strategies used by natural proteins are beyond our current abilities to constrain by design, but one possibility stands out as especially useful: a single charged side chain near the middle of what would ordinarily be the hydrophobic side of the edge beta strand. This minimal negative-design strategy changes only one residue, requires no backbone distortion, and is easy to design. The accompanying paper [Wang, W. & Hecht, M. H. (2002) Proc. Natl. Acad. Sci. USA 99, 2760-2765] makes use of the inward-pointing charge strategy with great success, turning highly aggregated beta-sandwich designs into soluble monomers.

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Unfolding of beta-sheet proteins in SDS.

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Unfolding of beta-sheet proteins in SDS.

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