Application Notes

4 each membrane protein a large number of different detergents have to be screened. However, limited supply due to low expression yields and low purification efficiency necessitate a simple and fast screening method, which requires only minor amounts of protein. In this study, we used the ion channel protein TehA from Haemophilus influenzae (HiTehA) as a test membrane protein to screen a number of different detergents to analyze their influence on thermal protein stability. For this, 10 µl aliquots of purified HiTehA in purification buffer (50 mM Tris HCl pH 8.0, 200 mM NaCl, 0.02 % DDM) at a concentration of 1.2 mg/ml was first diluted 1:2 into buffers containing an excess of different detergents to promote detergent exchange at the protein (Table 1). Next, a detergent-removal- column-based detergent exchange was performed, in which HiTehA was eluted into 20 µl of buffer with the respective detergent at the indicated final concentrations (Table 1). The final concentration of HiTehA for thermal unfolding experiments was 100 µg/ml, corresponding to a molar concentration of ~2 µM. The HiTehA solutions were loaded into nanoDSF grade capillaries and subjected to thermal unfolding using the Prometheus NT.48. HiTehA stability was analyzed in presence of 22 different detergents (Table 1) in duplicate experiments within a single run. Plots of the tryptophan fluorescence ratio (F350 nm / F330 nm), which detects both, changes in tryptophan intensity and emission peak position, show a clear unfolding transition of HiTehA under all conditions tested. Since the analysis of the fluorescence ratio at 350 and 330 nm selectively monitors shifts in tryptophan fluorescence, this approach is basically unaffected by unspecific autofluorescence of the detergents. A selection of thermal unfolding curves is shown in Figure 2A. Notably, detergent-dependent unfolding transition temperatures (Tm) differed by as much as 30°C. HiTehA was most stable in presence of the pyranoside-based detergents DDM (Tm = 62.8 ± 0.3 °C), DDTM (Tm = 61.37 ± 0.05 °C) and DdαM (Tm = 61.9 ± 0.05 °C), as well as in presence of the FOS-CHOLINEs FC-U10-11 (Tm = 59.2 ± 1.2 °C), FC-I11 (Tm = 58.5 ± 1.0 °C) and FC12 (Tm = 58.5 ± 1.6 °C). Conversely, several detergents such as C10E5 and C13E8 severely destabilized HiTehA, resulting in unfolding transition temperatures of 30.69 ± 0.24 °C and 33.17 ± 0.15 °C, respectively. Figure 3: Concentration dependence of HiTehA thermal stability. A) Initial discovery scan shows a broad range of fluorescence intensities prior to the thermal unfolding experiment. B) Thermal unfolding curves of HiTehA at different protein concentrations. C) Dependence of Tm of HiTehA on the total protein concentration.

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