(40f) Toward Controlled Conformational Change of Self-Assembled Vault Nanocapsules in Solution
Vaults are self-assembled ribonucleoprotein nanocapsules found in nearly all eukaryotic cells that consist of multiple copies of an untranslated RNA and multiple copies of 3 proteins (major vault protein, VPARP, and TEP1). The 96 copies of major vault protein (MVP) account for about 70% by mass of the entire vault particle, and stable vault particles can self-assemble from MVP alone in solution. Vault particles have approximate dimensions of 42 by 75 nm, and an apparent molecular weight of 13 x 106 daltons. The hollow interior volume is about 5 x 104 nm3 and is large enough to accommodate two ribosomes. Previous studies have shown that vaults dissociate into halves that open further into two flower-like structures on polylysine-coated mica surfaces. Our goal is to discover methods for reversible vault assembly/disassembly thereby enabling application of these nanocapsules in drug delivery and encapsulated materials synthesis.
Multi-angle laser light scattering (MALLS) was used to investigate the conformational changes of recombinant vaults in solution. These recombinant (CP-MVP) vaults contain 96 copies of MVP modified at the N-terminus with a 12 amino acid peptide from the metal binding protein, metallothionine, which contains 4 cysteine residues. Scattered light intensity data for vault preparations was analyzed using Zimm plots to obtain estimates of apparent molecular weight and radius of gyration. The radius of gyration was found to increase and molecular weight decrease as solution pH was shifted from 6.5 to 3.4, suggesting the disassembly of whole vaults into halves which open into flatter structures. Negatively stained transmission electron microscopy (TEM) images of vaults at pH 6.5 and 3.4 support the interpretation of MALLS data.
In addition to the dramatic conformational changes of vaults in response to pH, vault structure was found to be dynamic in neutral pH solution as well. In fact, relatively large proteins of 100 kDa or more have ready access to the vault interior. We have proposed the use of sulfhydryl cross-linking agents (e.g., BMH (bismaleimidohexane), BMOE (bis-maleimidoethane), and homobifunctional maleimide-PEG) to link the cysteine residues of CP-MVP vaults to give a more rigid structure. Based on the interpretation of MALLS data and TEM image analysis, cross-linked CP-MVP vaults appeared more stable. However, no whole vaults are observed when cross-linked vaults are exposed to pH 3.4 condition. Additional work is underway to cross-link reversibly the vault halves such that these nanocapsules may be used effectively as drug delivery packages or as compartments for nanomaterial synthesis.
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