Spectroscopic Characterization of Structural Changes in Membrane Scaffold Proteins Entrapped within Mesoporous Silica Gel Monoliths.

ABSTRACT

The changes in the orientation and conformation of three different membrane scaffold proteins (MSPs) upon entrapment in sol-gel-derived mesoporous silica monoliths were investigated. MSPs were examined in either a lipid-free or a lipid-bound conformation, where the proteins were associated with lipids to form nanolipoprotein particles (NLPs). NLPs are water-soluble, disk-shaped patches of a lipid bilayer that have amphiphilic MSPs shielding the hydrophobic lipid tails. The NLPs in this work had an average thickness of 5 nm and diameters of 9.2, 9.7, and 14.8 nm. We have previously demonstrated that NLPs are more suitable lipid-based structures for silica gel entrapment than liposomes because of their size compatibility with the mesoporous network (2-50 nm) and minimally altered structure after encapsulation. Here we further elaborate on that work by using a variety of spectroscopic techniques to elucidate whether or not different MSPs maintain their protein-lipid interactions after encapsulation. Fluorescence spectroscopy and quenching of the tryptophan residues with acrylamide, 5-DOXYL-stearic acid, and 16-DOXYL-stearic acid were used to determine the MSP orientation. We also utilized fluorescence anisotropy of tryptophans to measure the relative size of the NLPs and MSP aggregates after entrapment. Finally, circular dichroism spectroscopy was used to examine the secondary structure of the MSPs. Our results showed that, after entrapment, all of the lipid-bound MSPs maintained orientations that were minimally changed and indicative of association with lipids in NLPs. The tryptophan residues appeared to remain buried within the hydrophobic core of the lipid tails in the NLPs and appropriately spaced from the bilayer center. Also, after entrapment, lipid-bound MSPs maintained a high degree of α-helical content, a secondary structure associated with protein-lipid interactions. These findings demonstrate that NLPs are capable of serving as viable hosts for functional integral membrane proteins in the synthesis of sol-gel-derived bioinorganic hybrid nanomaterials.