BIOTECHNOLOGY
Structural Stability of Vault Particles

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ABSTRACT

Vaults, at 13 MDa, are the largest ribonucleoprotein particles known. In vitro, expression of the major vault protein (MVP) alone in Sf9 insect cells results in the production of recombinant particles with characteristic vault structure. With the ultimate goal of using recombinant vaults as nanocapsules for the delivery of biomolecules, we have employed a variety of spectroscopic techniques (i.e., circular dichroism, fluorescence spectroscopy, and light scattering) along with electron microscopy, to characterize the structural stability of vaults over a wide range of pH (3–8) and temperature (10–90°C). Ten different conformational states of the vaults were identified over the pH and temperature range studied with the most stable region at pH 6–8 below 40°C and least stable at pH 4–6 above 60°C. A unique intermediate molten globulelike state was also identified at pH 6 and ~ 55°C. EM imaging showed the opening of intact vaults into flowerlike structures when transitioning from neutral to acidic pH. This information has potential use in the development of recombinant vaults into nanocapsules for drug delivery since one mechanism by which therapeutic agents entrapped in vaults could be released is through an opening of the intact vault structure.

Section snippets

INTRODUCTION

Some 20 years ago, Kedersha and Rome purified a large ribonucleoprotein particle from rat liver homogenates and named it the “vault” particle on the basis of its morphological resemblance to vaulted ceilings in medieval cathedrals.1., 2. Cryoelectron microscopy (CryoEM) single particle reconstructions and X-ray crystallography show vaults to posses a hollow, barrellike structure with two protruding caps and an invaginated waist.3., 4. With a molecular weight of 12.9 ± 1 MDa and dimensions of ~ 420 Å

Vault Purification

Recombinant vaults formed from CP-MVP were purified from baculovirus infected Sf9 cells, as previously described.5., 13. A 12-residue cysteine rich motif (MAGCGCPCGCGA) on the N-terminus of CP-MVP was previously shown to help stabilize the recombinant vaults.4., 13. Purified vault particles were resuspended in 20 mM citrate-phosphate buffer pH 6.5.

Sample Preparation

Vault solutions at different pH values were prepared in 20 mM isotonic citrate/phosphate buffer by dialyzing stock solutions into buffers ranging from

Far-UV Circular Dichroism (CD) Spectroscopy

Alterations in vault secondary structure were studied by monitoring the changes in the CD signal over a wide range of pH (3–8) when subjected to thermal gradients from 10 to 90°C. The CD signal is expressed in units of mean residue ellipticity which are independent of molecular weight, since the average molecular weight of vault particles in solution vary across the pH range due to conformational alterations. Using MALLS, Goldsmith et al.14 showed that the average molecular weight of CP-MVP

DISCUSSION

Employing a variety of techniques, we have extensively characterized the behavior of vaults over a wide range of solution variables (i.e., pH and temperature). The pH range examined covers the extent of physiological pH in different cellular compartments and organs and should therefore provide a basis for further interpretation of vault behavior in vivo. In addition, the combination of pH and temperature effects on the structural integrity of vaults should aid in formulation of these

CONCLUSION

Understanding the effects of pH and temperature on vault stability should aid in the ultimate goal of utilizing these particles as potential drug delivery devices. Our studies show that vault conformation was altered as a function of decreasing pH in which intact particles at neutral pH open into flowerlike structures at acidic pH. Opening of intact vaults could be potentially developed as an intracellular controlled-release device for drug delivery.

With the identification of at least 10

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