Role of nonionic surfactants in promoting the folding and stability of integral membrane proteins

Membrane proteins are characterized by their extensive hydrophobic surface area designed to interact with the lipid bilayer. These proteins provide functionality to the cell membrane by stabilizing this interface, acting as selective transporters and receptors. Because of their central role in communications between the cell and its environment, membrane proteins are common targets for pharmaceuticals.;Nonionic surfactants are commonly used amphiphiles that can serve to shield the hydrophobic surface area of membrane proteins and allow their solubilization in aqueous solutions, resulting in the formation of protein-detergent complexes. The ideal surfactant is able to maintain the physiological function and structure of a target membrane protein in solution, therefore preventing unfolding and aggregation. Currently, trial and error is necessary to determine the best nonionic surfactant for each membrane protein target.;This thesis focuses on the role of nonionic surfactants in the folding and stability of integral membrane proteins. Two points of view are considered: the role of the surfactant in establishing and stabilizing the protein structure, and the morphology and interactions of protein-detergent complexes. The main integral membrane protein under consideration was the outer membrane protein X, ompX, from E. coli, and its interactions were studied with polyoxyethylene nonionic surfactants noctyltetraoxyethylene (C8 E4), n-octylpentaoxyethylene, (C8E5), n-dodecylhexaoxyethylene (C12E6) and n-dodecyloctaoxyethylene (C12E8).;OmpX production was achieved through recombinant inclusion body expression in its native host, followed by purification in a denatured conformation. A rapid dilution refolding protocol was developed and was applied to compare the refolding of ompX in various nonionic surfactants. The final protein structure was determined from circular dichroism, fluorescence and FTIR spectroscopy analyses. The secondary structure of ompX was found to be very similar in all surfactants studied, and comparable to the estimates from its NMR structure and from the secondary structure of other refolded outer membrane proteins.;Nonionic surfactants self-assemble into micelles in aqueous solutions at concentrations above the critical micelle concentration (CMC). Solutions of nonionic surfactants at concentrations above their CMC may display a phase separation with increasing temperature. The lowest temperature at which this separation can be detected is called the cloud point temperature (CPT). As the CPT is approached, micelle interactions can be considered to become more attractive or their morphology to become more elongated.;In the process of understanding how surfactants can stabilize integral membrane proteins, it was necessary to examine how the chaotropic agent urea, commonly used in protein unfolding studies, affects surfactant phase behavior. Urea was found to increase the CMC of all polyoxyethylene surfactants studied significantly, as well as increase their CPT. The effect of urea on micelle morphology and interaction was probed by small-angle neutron scattering (SANS) experiments. It was found that micelles of C8E4 and C12E6 appeared elongated without urea in solution and become globular with increasing urea concentration. This effect was absent for C8E5 and C12E8 micelles, which remained globular independent of urea concentration. Therefore, we suggest an analogy between the effect of increasing urea concentration and decreasing temperature on the phase behavior of polyoxyethylene surfactants.;The ability of nonionic surfactants to stabilize ompX in solution was tested by unfolding the PDC by increasing the urea concentration. Loss of protein structure was monitored by circular dichroism and fluorescence spectroscopies. Surfactant type and concentration were found to influence ompX unfolding. The protein stability was greatly diminished if the surfactant concentration fell below the CMC compared to when the surfactant concentration was adjusted to account for CMC changes with increasing urea concentration.;The interactions and morphologies of ompX PDCs in solution with micelles of C8E4, C8E5, C12E 6 and C12E8 surfactants with increasing urea concentration were examined by SANS. The challenge of these experiments was to distinguish the scattering of PDCs from that of micelles. Differences between the two sets of scattering curves are most prominent at urea concentrations below 2 M, and become small at urea concentrations at which ompX is denatured. These differences are more prominent in C8 surfactants at 0 and 2 M urea, which contain elongated particles interpreted as aggregated PDCs. Simple models were applied to analyze the scattering of solutions containing PDCs and micelles but were unable to capture the entire scattering curves within physically realistic parameter constraints.