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
