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Abstract While α‐synuclein, an intrinsically disordered protein linked to Parkinson's disease, has been shown to associate with membrane organelles, its overall cellular function remains nebulous. α‐Synuclein binds to membranes through its amino‐terminal domain (first ≈100 residues), but there is no consensus on the biophysical function of the carboxyl‐terminal domain (last ≈40 residues) due, in part, to its lack of strong interaction partners and persisting intrinsic disorder even when membrane bound. Here, by directly applying force on α‐synuclein bound to spherical nanoparticle‐supported lipid bilayers (SSLBs) and tracking higher‐order structural changes through small‐angle X‐ray scattering, strong evidence is presented that α‐synuclein sterically stabilizes membrane surfaces through its carboxyl‐terminal domain. Full‐length α‐synuclein dramatically increases the critical osmotic pressure at which SSLBs cluster (PC≈ 1.3 × 105Pa) compared to α‐synuclein without the carboxyl‐terminal domain (PC≈ 1.9 × 104Pa) at physiological salt and temperature conditions. This clustering of α‐synuclein‐bound SSLBs is shown to be reversible and sensitive to monovalent/divalent salt, both features of grafted polyelectrolyte‐mediated steric stabilization. In elucidating the biophysical function of α‐synuclein in the framework of polymer science, it is demonstrated that the carboxyl‐terminal domain can potentially utilize its persisting intrinsic disorder to functionalize membrane surfaces.
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One‐step site‐specific S ‐alkylation of full‐length caveolin‐1: Lipidation modulates the topology of its C ‐terminal domain
Abstract Caveolin‐1 is an integral membrane protein that is known to acquire a number of posttranslational modifications upon trafficking to the plasma membrane. In particular, caveolin‐1 is palmitoylated at three cysteine residues (C133, C143, and C156) located within theC‐terminal domain of the protein which could have structural and topological implications. Herein, a reliable preparation of full‐lengthS‐alkylated caveolin‐1, which closely mimics the palmitoylation observed in vivo, is described. HPLC and ESI‐LC‐MS analyses verified the addition of the C16 alkyl groups to caveolin‐1 constructs containing one (C133), two (C133 and C143), and three (C133, C143, and C156) cysteine residues. Circular dichroism spectroscopy analysis of the constructs revealed thatS‐alkylation does not significantly affect theglobalhelicity of the protein; however, molecular dynamics simulations revealed that there werelocalregions where the helicity was altered positively or negatively byS‐alkylation. In addition, the simulations showed that lipidation tames the topological promiscuity of theC‐terminal domain, resulting in a disposition within the bilayer characterized by increased depth.
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- Award ID(s):
- 2111728
- PAR ID:
- 10470719
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Protein Science
- Volume:
- 32
- Issue:
- 11
- ISSN:
- 0961-8368
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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