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|Title:||Understanding the interaction between major vault protein and INT for application in modulated molecular release||Authors:||Yu, Kang||Keywords:||DRNTU::Engineering::Bioengineering||Issue Date:||2014||Abstract:||The vault, a ribonucleoprotein nanoparticle, is ubiquitous in most living eukaryotic systems with dimensions of 72.5 × 41.0 nm. It has been shown to be promising as a carrier for small molecule drugs. The nature-derived template provides precise spatial control resulting in highly uniform structures while the proteinaceous nature allows for facile modification. Vaults are non-toxic, non-immunogenic, and stable over wide pH and temperature ranges. The vault shell is made of multiple copies of the major vault protein (MVP) that self-assemble to form a barrel-like nanocapsule. A shuttle peptide, referred to as the interaction domain (INT) located at the C-terminus of the vault poly(ADP-ribose)-polymerase (GenBank accession No. AF158255; aa 1563-1724), is known to attach to the inner side of the vault shell through a unique protein-protein interaction. Fusion of therapeutic agents to the INT N-terminus facilitates the encapsulation of therapeutic agents within the vault. Introduction of some C-terminal peptide extensions to the MVP were used to target vaults to the cell surface receptor. The vault has a dynamic structure that involves breathing and half-vault exchange. During the open state, content might be freed from or held within the lumen depending on the association/dissociation between MVP and INT. However, modulating the release of therapeutic agents from the vaults interior remains a challenge. In this study, we seek to understand the interactions between the MVP and INT by determining the binding constants using surface plasmon resonance (SPR) technology, modifying the interactions by histidine substitution, and characterizing the physicochemical properties. To facilitate purification, all studies have been performed on N-terminal hexa-histidine fusion proteins. Specific domains on the MVP that interact with INT (domains 3, 4 and 5) are identified using protein docking software and are referred to as iMVP (PDB ID 2QZV, aa 102-276). The binding between His-iMVP and His-INT has been subsequently determined to be 1:1 at His-iMVP immobilization density of 410 RU with affinity constant KD of 262 nM. To tune the release of molecular cargos from the vault nanoparticles, the protein-protein interaction was modified by introduction of multiple histidines to replace key amino acids on the INT located at the interaction interface. The histidine-substituted His-INT showed stronger interaction with His-iMVP than wild type His-INT at pH 6.0 - 7.4. It was hypothesized that the rate of molecular release from the vault lumen could be tuned by mixing the wild type and histidine-substituted His-INT. The equimolar mixture displayed intermediate affinity constant at pH 6.0 and 7.4. For application in drug delivery, the stability of His-INT variants was studied by monitoring the proteins unfolding and aggregation. The melting temperature of wild type His-INT reached as high as 67°C in 20 mM PBS at pH 6.0 and 7.0 in the presence of 150 mM NaCl while aggregation was observed starting at 42°C. At NaCl concentration of 150 mM, the proteins at low pH aggregated later than at high pH. However, the unfolding occurred relatively sooner. The histidine-substitution on His-INT resulted in conversion from α-helix to random coil and reduced protein stability.||URI:||http://hdl.handle.net/10356/55681||Fulltext Permission:||restricted||Fulltext Availability:||With Fulltext|
|Appears in Collections:||SCBE Theses|
checked on Sep 28, 2020
checked on Sep 28, 2020
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