Bioengineering Division, School of Chemical and Biomedical Engineering, Nanyang Technological University, 70 Nanyang Drive, Singapore, 637457, Singapore.
Structural Biology and Biochemistry Division, School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore, 637551, Singapore.
Sci Rep. 2017 Nov 1;7(1):14816. doi: 10.1038/s41598-017-12870-x.
Vaults are naturally occurring ovoid nanoparticles constructed from a protein shell that is composed of multiple copies of major vault protein (MVP). The vault-interacting domain of vault poly(ADP-ribose)-polymerase (INT) has been used as a shuttle to pack biomolecular cargo in the vault lumen. However, the interaction between INT and MVP is poorly understood. It is hypothesized that the release rate of biomolecular cargo from the vault lumen is related to the interaction between MVP and INT. To tune the release of molecular cargos from the vault nanoparticles, we determined the interactions between the isolated INT-interacting MVP domains (iMVP) and wild-type INT and compared them to two structurally modified INT: 15-amino acid deletion at the C terminus (INTΔC15) and histidine substituted at the interaction surface (INT/DSA/3 H) to impart a pH-sensitive response. The apparent affinity constants determined using surface plasmon resonance (SPR) biosensor technology are 262 ± 4 nM for iMVP/INT, 1800 ± 160 nM for iMVP/INTΔC15 at pH 7.4. The INT/DSA/3 H exhibits stronger affinity to iMVP (K = 24 nM) and dissociates at a slower rate than wild-type INT at pH 6.0.
穹窿是由一种蛋白壳构成的天然存在的椭圆形纳米颗粒，该蛋白壳由多个主要穹窿蛋白（MVP）拷贝组成。穹窿-多聚（ADP-核糖）-聚合酶的穹窿相互作用域（INT）已被用作穿梭蛋白，将生物分子货物包装在穹窿腔中。然而，INT 与 MVP 之间的相互作用仍不清楚。据推测，生物分子货物从穹窿腔中的释放速率与 MVP 和 INT 之间的相互作用有关。为了调节从穹窿纳米颗粒中释放分子货物的速度，我们确定了分离的 INT 相互作用 MVP 结构域（iMVP）与野生型 INT 之间的相互作用，并将其与两种结构修饰的 INT 进行了比较：C 端 15 个氨基酸缺失（INTΔC15）和相互作用表面的组氨酸取代（INT/DSA/3H）以赋予 pH 敏感响应。使用表面等离子体共振（SPR）生物传感器技术确定的表观亲和力常数分别为 262±4 nM 用于 iMVP/INT，1800±160 nM 用于 iMVP/INTΔC15 在 pH 7.4 下。INT/DSA/3H 与 iMVP 的亲和力更强（K = 24 nM），并且在 pH 6.0 下比野生型 INT 以更慢的速度解离。
