These scaffolds provide a rich set of starting points for binding a wide range of C2 symmetric compounds.
Of these, the geometry of 31 were confirmed by small angle X-ray scattering and 2 were shown by crystallographic analyses to be in close agreement with the computational design models. We used this approach to design thousands of C2 symmetric homodimers, and characterized 101 of them experimentally. We first designed repeat proteins that sample a continuum of curvatures but have low helical rise, then docked these into C2 symmetric homodimers to generate an extensive range of C2 symmetric cavities. Here, we describe a general approach to designing hyperstable C2 symmetric proteins with pockets of diverse size and shape. For design of binding to symmetric ligands, protein homo-oligomers with matching symmetry are advantageous as each protein subunit can make identical interactions with the ligand. This work demonstrates that antigen-displaying protein nanoparticles can be designed from scratch, and provides a systematic way to investigate the influence of antigen presentation geometry on the immune response to vaccination.įunction follows form in biology, and the binding of small molecules requires proteins with pockets that match the shape of the ligand. Electron microscopy and antibody binding experiments demonstrated that the designed nanoparticles presented antigenically intact prefusion HIV-1 Env, influenza hemagglutinin, and RSV F trimers in the predicted geometries. Trimers that experimentally adopted their designed configurations were incorporated as components of tetrahedral, octahedral, and icosahedral nanoparticles, which were characterized by cryo-electron microscopy and assessed for their ability to present viral glycoproteins. We first de novo designed trimers tailored for antigen fusion, featuring N-terminal helices positioned to match the C termini of the viral glycoproteins. To enable a new generation of anti-viral vaccines, we designed self-assembling protein nanoparticles with geometries tailored to present the ectodomains of influenza, HIV, and RSV viral glycoprotein trimers. In our view, major advantages of nanodisc technology for integral membrane proteins is homogeneity, control of oligomerization state, access to both sides of the membrane, and control of lipids in the local membrane environment of the integral protein.Multivalent presentation of viral glycoproteins can substantially increase the elicitation of antigen-specific antibodies. In the present review, we outline the biological inspiration for nanodiscs as discoidal high-density lipoproteins, the assembly and handling of nanodiscs, and finally their diverse biochemical applications. After reconstitution, the membrane nanodisc is soluble, stable, and monodisperse.
A developing technology termed nanodiscs exploits discoidal phospholipid bilayers encircled by a stabilizing amphipatic helical membrane scaffold protein to reconstitute membranes with integral proteins. However, solubilization in detergents or reconstitution in liposomes or supported monolayers sometimes suffers from loss of activity and problematic analyses due to heterogeneity and aggregation. This requires the incorporation of the membrane proteins into a native-like membrane or detergent micelle that mimics the properties of the original biological membrane. A major challenge in the research on membrane-anchored and integral membrane protein complexes is to obtain these in a functionally active, water-soluble, and monodisperse form.
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