Interactions of Fluorinated Surfactants with Diphtheria Toxin T-Domain: Testing New Media for Studies of Membrane Proteins

Abstract

The principal difficulty in experimental exploration of the folding and stability of membrane proteins (MPs) is their aggregation outside of the native environment of the lipid bilayer. To circumvent this problem, we recently applied fluorinated nondetergent surfactants that act as chemical chaperones. The ideal chaperone surfactant would 1), maintain the MP in solution; 2), minimally perturb the MP's structure; 3), dissociate from the MP during membrane insertion; and 4), not partition into the lipid bilayer. Here, we compare how surfactants with hemifluorinated (HFTAC) and completely fluorinated (FTAC) hydrophobic chains of different length compare to this ideal. Using fluorescence correlation spectroscopy of dye-labeled FTAC and HFTAC, we demonstrate that neither type of surfactant will bind lipid vesicles. Thus, unlike detergents, fluorinated surfactants do not compromise vesicle integrity even at concentrations far in excess of their critical micelle concentration. We examined the interaction of surfactants with a model MP, DTT, using a variety of spectroscopic techniques. Site-selective labeling of DTT with fluorescent dyes indicates that the surfactants do not interact with DTT uniformly, instead concentrating in the most hydrophobic patches. Circular dichroism measurements suggest that the presence of surfactants does not alter the structure of DTT. However, the cooperativity of the thermal unfolding transition is reduced by the presence of surfactants, especially above the critical micelle concentration (a feature of regular detergents, too). The linear dependence of DTT's enthalpy of unfolding on the surfactant concentration is encouraging for future application of (H)FTACs to determine the stability of the membrane-competent conformations of other MPs. The observed reduction in the efficiency of Förster resonance energy transfer between donor-labeled (H)FTACs and acceptor-labeled DTT upon addition of lipid vesicles indicates that the protein sheds the layer of surfactant during its bilayer insertion. We discuss the advantages of fluorinated surfactants over other types of solubilizing agents, with a specific emphasis on their possible applications in thermodynamic measurements.

Detergent-activated BAX Protein Is a Monomer

“…The reason for such variation of the fluorescence brightness of the protein monomer in various detergents was because of the enhancement or quenching of the Bodipy FL fluorophore brightness upon transfer into the hydrophobic environment of a detergent micelle. Similar effects of the fluorophore brightness enhancement were reported previously for the fluorescently labeled diphtheria toxin T-domain interacting with detergent micelles (34). In our case there appears to be an enhancement of the fluorescence brightness of fluor-BAX⌬C in the presence of n-octyl glucoside, dodecyl maltoside, and Triton X-100 detergents, and a decrease in protein fluorescence brightness in the presence of cholic acid and Tween 20.…”

Calibration of Distribution Analysis of the Depth of Membrane Penetration Using Simulations and Depth-Dependent Fluorescence Quenching

“…In past years we have used a series of NBD-labeled single-cysteine mutants of the T-domain of diphtheria toxin to study its acid-induced binding and insertion into lipid membranes (Ladokhin et al 2004), (Kyrychenko et al 2009; Rodnin et al 2008; Rodnin et al 2010). The T-domain has two hydrophobic α-helices TH8 and 9, which form a transmembrane hairpin.…”

“…For determination of protein concentrations we used a molar extinction coefficient of 17000 M −1 cm −1 at 278 nm. Labeling with NBD dye was performed using a standard procedure for the thiol-reactive derivatives (Rodnin et al 2008; Kyrychenko et al 2009). …”

Diphtheria toxin

“…The general fold and the secondary structure are maintained, but packing is loose, tertiary constraints are lost, and hydrophobic surfaces of the core of the protein are accessible. As a consequence, the T domain is prone to bind and penetrate a phospholipid bilayer [44,[109][110][111]. His223, His257, and His251 are the most sensitive triggers for the formation of the molten globule state in solution, whereas His322, His323, and His251 are the most sensitive triggers for membrane binding [112].…”