Solubilizing membrane proteins for functional, structural and thermodynamic studies is usually achieved with the help of detergents, which tend to destabilize them, however. Several classes of non-detergent surfactants have been designed as milder substitutes for detergents, most prominently amphipathic polymers called 'amphipols' and fluorinated surfactants. Here we test the potential usefulness of these compounds for thermodynamic studies by examining their effect on conformational transitions of the diphtheria toxin T-domain. The advantage of the T-domain as a model system is that it exists as a soluble globular protein at neutral pH yet is converted into a membrane-competent form by acidification and inserts into the lipid bilayer as part of its physiological action. We have examined the effects of various surfactants on two conformational transitions of the T-domain, thermal unfolding and pH-induced transition to a membrane-competent form. All tested detergent and non-detergent surfactants lowered the cooperativity of the thermal unfolding of the T-domain. The dependence of enthalpy of unfolding on surfactant concentration was found to be least for fluorinated surfactants, thus making them useful candidates for thermodynamic studies. Circular dichroism measurements demonstrate that non-ionic homo-polymeric amphipols (NAhPols), unlike any other surfactants, can actively cause a conformational change of the T-domain. NAhPol-induced structural rearrangements are different from those observed during thermal denaturation and are suggested to be related to the formation of the membrane-competent form of the T-domain. Measurements of vesicle content leakage indicate that interaction with NAhPols not only does not prevent the T-domain from inserting into the bilayer, but it can make bilayer permeabilization even more efficient, whereas the pH-dependence of membrane permeabilization becomes more cooperative.

To prepare non-ionic amphiphilic polymers usable for solubilizing integral membrane proteins, hydrophilic and amphiphilic β-D-glucose-based tris(hydroxymethyl)acrylamidomethane monomers were synthesized and their radical homopolymerization rate constants (f k 2 p /k t ) were determined. The polymerization kinetics, monitored by 1 H-NMR, were carried out in THF in the presence of AIBN as radical initiator. The plot of monomer conversion as a function of time followed Tobolsky's equation. The number of hydroxyl groups, as well as the nature of the substituents grafted onto the hydroxyl groups, was found to alter the rates of polymerization. While the steric hindrance significantly affected the kinetics, intermolecular hydrogen bonds between hydroxyl groups, as demonstrated by NMR investigations at different concentrations and temperatures, may also play a role in the rate constants of polymerization.