Reversible Unfolding of Rhomboid Intramembrane Proteases.

Denaturant-induced unfolding of helical membrane proteins provides insights into their mechanism of folding and domain organization, which take place in the chemically heterogeneous, anisotropic environment of a lipid membrane. Rhomboid proteases are intramembrane proteases that play key roles in various diseases. Crystal structures have revealed a compact helical bundle with a buried active site, which requires conformational changes for the cleavage of transmembrane substrates. A dimeric form of the rhomboid protease has been shown to be important for activity. In this study, we examine the mechanism of refolding for two distinct rhomboids to gain insight into their secondary structure-activity relationships. Although helicity is largely abolished in the unfolded states of both proteins, unfolding is completely reversible for HiGlpG but only partially reversible for PsAarA. Refolding of both proteins results in reassociation of the dimer, with a 90% regain of catalytic activity for HiGlpG but only a 70% regain for PsAarA. For both proteins, a broad, gradual transition from the native, folded state to the denatured, partly unfolded state was revealed with the aid of circular dichroism spectroscopy as a function of denaturant concentration, thus arguing against a classical two-state model as found for many globular soluble proteins. Thermal denaturation has irreversible destabilizing effects on both proteins, yet reveals important functional details regarding substrate accessibility to the buried active site. This concerted biophysical and functional analysis demonstrates that HiGlpG, with a simple six-transmembrane-segment organization, is more robust than PsAarA, which has seven predicted transmembrane segments, thus rendering HiGlpG amenable to in vitro studies of membrane-protein folding.

Characterising protein/detergent complexes by triple-detection size-exclusion chromatography.

BACKGROUND: In vitro investigations of membrane proteins usually depend on detergents for protein solubilisation and stabilisation. The amount of detergent bound to a membrane protein is relevant to successful experiment design and data analysis but is often unknown. Triple-detection size-exclusion chromatography enables simultaneous separation of protein/detergent complexes and protein-free detergent micelles and determination of their molar masses in a straightforward and absolute manner. Size-exclusion chromatography is used to separate different species, while ultraviolet absorbance, static light scattering, and refractive index measurements allow molar mass determination of protein and detergent components. RESULTS: We refined standard experimental and data-analysis procedures for challenging membrane-protein samples that elude routine approaches. The general procedures including preparatory steps, measurements, and data analysis for the characterisation of both routine and complex samples in difficult solvents such as concentrated denaturant solutions are demonstrated. The applicability of the protocol but also its limitations and possible solutions are discussed, and an extensive troubleshooting section is provided. CONCLUSIONS: We established and validated a protocol for triple-detection size-exclusion chromatography that enables the inexperienced user to perform and analyse measurements of well-behaved protein/detergent complexes. More experienced users are provided with an example of a more sophisticated analysis procedure allowing mass determination under challenging separation conditions.

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 ß-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.