Impact of Urea on Detergent Micelle Properties

Co-solvents, such as urea, can entail drastic changes in the micellization behavior of detergents. We present a systematic quantification of the impact of urea on the critical micellar concentration, the micellization thermodynamics, and the micelle size in three homologous series of commonly used non-ionic alkyl detergents. To this end, we performed demicellization experiments by isothermal titration calorimetry and hydrodynamic size measurements by dynamic light scattering on alkyl maltopyranosides, cyclohexyl alkyl maltopyranosides, and alkyl glucopyranosides at urea concentrations of 0–8 M. For all detergents studied, we found that the critical micellar concentration increases exponentially because the absolute Gibbs free energy of micellization decreases linearly over the entire urea concentration range, as does the micelle size. In contrast, the enthalpic and entropic contributions to micellization reveal more complex, nonlinear dependences on urea concentration. Both free energy and size changes are more pronounced for long-chain detergents, which bury more apolar surface area upon micelle formation. The Gibbs free energy increments per methylene group within each detergent series depend on urea concentration in a linear fashion, although they result from the entropic term for alkyl maltosides but are of enthalpic origin for cyclohexyl alkyl maltosides. We compare our results to transfer free energies of amino acid side chains, relate them to protein-folding data, and discuss how urea-induced changes in detergent micelle properties affect <i>in vitro</i> investigations on membrane proteins

Polar Interactions Trump Hydrophobicity in Stabilizing the Self-Inserting Membrane Protein Mistic

Canonical integral membrane proteins are attached to lipid bilayers through hydrophobic transmembrane helices, whose topogenesis requires sophisticated insertion machineries. By contrast, membrane proteins that, for evolutionary or functional reasons, cannot rely on these machineries need to resort to driving forces other than hydrophobicity. A striking example is the self-inserting Bacillus subtilis protein Mistic, which is involved in biofilm formation and has found application as a fusion tag supporting the recombinant production and bilayer insertion of other membrane proteins. Although this unusual protein contains numerous polar and charged residues and lacks characteristic membrane-interaction motifs, it is tightly bound to membranes in vivo and membrane-mimetic systems in vitro. Therefore, we set out to quantify the contributions from polar and nonpolar interactions to the coupled folding and insertion of Mistic. To this end, we defined conditions under which the protein can be unfolded completely and reversibly from various detergent micelles by urea in a two-state equilibrium and where the unfolded state is independent of the detergent used for solubilizing the folded state. This enabled equilibrium unfolding experiments previously used for soluble and β-barrel membrane proteins, revealing that polar interactions with ionic and zwitterionic headgroups and, presumably, the interfacial dipole potential stabilize the protein much more efficiently than nonpolar interactions with the micelle core. These findings unveil the forces that allow a protein to tightly interact with a membrane-mimetic environment without major hydrophobic contributions and rationalize the differential suitability of detergents for the extraction and solubilization of Mistic-tagged membrane proteins

A Versatile System for High-Throughput In Situ X‑ray Screening and Data Collection of Soluble and Membrane-Protein Crystals

In recent years, in situ data collection has been a major focus of progress in protein crystallography. Here, we introduce the Mylar in situ method using Mylar-based sandwich plates that are inexpensive, easy to make and handle, and show significantly less background scattering than other setups. A variety of cognate holders for patches of Mylar in situ sandwich films corresponding to one or more wells makes the method robust and versatile, allows for storage and shipping of entire wells, and enables automated crystal imaging, screening, and goniometer-based X-ray diffraction data-collection at room temperature and under cryogenic conditions for soluble and membrane-protein crystals grown in or transferred to these plates. We validated the Mylar in situ method using crystals of the water-soluble proteins hen egg-white lysozyme and sperm whale myoglobin as well as the 7-transmembrane protein bacteriorhodopsin from Haloquadratum walsbyi. In conjunction with current developments at synchrotrons, this approach promises high-resolution structural studies of membrane proteins to become faster and more routine