Photoinitiated polymerization-induced
self-assembly (photo-PISA)
is an efficient approach to predictably prepare polymeric nanostructures
with a wide range of morphologies. Given that this process can be
performed at high concentrations and under mild reaction conditions,
it has the potential to have significant industrial scope. However,
given that the majority of industrial (and more specifically biotechnological)
formulations contain mixtures of polymers and surfactants, the effect
of such surfactants on the PISA process is an important consideration.
Thus, to expand the scope of the methodology, the effect of small
molecule surfactants on the PISA process, specifically for the preparation
of unilamellar vesicles, was investigated. Similar to aqueous photo-PISA
findings in the absence of surfactant molecules, the originally targeted
vesicular morphology was retained in the presence of varying concentrations
of non-ionic surfactants, while a diverse set of lower-order morphologies
was observed for ionic surfactants. Interestingly, a critical micelle
concentration (CMC)-dependent behavior was detected in the case of
zwitterionic detergents. Additionally, tunable size and membrane thickness
of vesicles were observed by using different types and concentration
of surfactants. Based on these findings, a functional channel-forming
membrane protein (OmpF porin), stabilized in aqueous media by surfactant
molecules, was able to be directly inserted into the membrane of vesicles
during photo-PISA. Our study demonstrates the potential of photo-PISA
for the direct formation of protein–polymer complexes and highlights
how this method could be used to design biomimicking polymer/surfactant
nanoreactors.
The interplay between membrane proteins and the lipids of the membrane is important for cellular function, however, tools enabling the interrogation of protein dynamics within native lipid environments are scarce and often invasive. We show that the styrene-maleic acid lipid particle (SMALP) technology can be coupled with hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate membrane protein conformational dynamics within native lipid bilayers. We demonstrate changes in accessibility and dynamics of the rhomboid protease GlpG, captured within three different native lipid compositions, and identify protein regions sensitive to changes in the native lipid environment. Our results illuminate the value of this approach for distinguishing the putative role(s) of the native lipid composition in modulating membrane protein conformational dynamics.
Understanding how an amino acid sequence folds into a functional, three-dimensional structure has proved to be a formidable challenge in biological research, especially for transmembrane proteins with multiple alpha helical domains. Mechanistic folding studies on helical membrane proteins have been limited to unusually stable, single domain proteins such as bacteriorhodopsin. Here, we extend such work to flexible, multidomain proteins and one of the most widespread membrane transporter families, the major facilitator superfamily, thus showing that more complex membrane proteins can be successfully refolded to recover native substrate binding. We determine the unfolding free energy of the two-domain, Escherichia coli galactose transporter, GalP; a bacterial homologue of human glucose transporters. GalP is reversibly unfolded by urea. Urea causes loss of substrate binding and a significant reduction in alpha helical content. Full recovery of helical structure and substrate binding occurs in dodecylmaltoside micelles, and the unfolding free energy can be determined. A linear dependence of this free energy on urea concentration allows the free energy of unfolding in the absence of urea to be determined as þ2.5 kcal·mol −1 . Urea has often been found to be a poor denaturant for transmembrane helical structures. We attribute the denaturation of GalP helices by urea to the dynamic nature of the transporter structure allowing denaturant access via the substrate binding pocket, as well as to helical structure that extends beyond the membrane. This study gives insight into the final, critical folding step involving recovery of ligand binding for a multidomain membrane transporter.protein folding | thermodynamic stability | linear free-energy relationship
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