Charge reduction stabilizes intact membrane protein complexes for mass spectrometry

Abstract

The study of intact soluble protein assemblies by means of mass spectrometry is providing invaluable contributions to structural biology and biochemistry. A recent breakthrough has enabled similar study of membrane protein complexes, following their release from detergent micelles in the gas phase. Careful optimization of mass spectrometry conditions, particularly with respect to energy regimes, is essential for maintaining compact folded states as detergent is removed. However, many of the saccharide detergents widely employed in structural biology can cause unfolding of membrane proteins in the gas phase. Here, we investigate the potential of charge reduction by introducing three membrane protein complexes from saccharide detergents and show how reducing their overall charge enables generation of compact states, as evidenced by ion mobility mass spectrometry. We find that charge reduction stabilizes the oligomeric state and enhances the stability of lipid-bound complexes. This finding is significant since maintaining native-like membrane proteins enables ligand binding to be assessed from a range of detergents that retain solubility while protecting the overall fold.

Figure 1

Charge reduction of the trimeric outer membrane protein OmpF. (a) Mass spectra recorded for a solution containing 200 mM ammonium acetate and 1% OG (standard conditions), (b) exposed to acetonitrile vapor, (c) 10 mM imidazole added in solution, and (d) exposed to acetonitrile with addition of imidazole. (e) Plot of average CCS measured for all charge states of OmpF. Theoretical CCS calculated from the PDB (3O0E) and for collapsed states derived from MD (red and green, respectively). (f) Arrival time distribution of the +16 charge state showing peak splitting coincident with CCS of native-like and partially collapsed structures (red and green, respectively). Inset: structure of OmpF highlighting its disc-like structure embedded in the micelle.

Figure 2

Charge reduction and its effect on the CCS of P-gp. Plot of the average CCS measured for different charge states before and after charge reduction as in Figure 1a–d. Inset spectra for cardiolipin binding to P-gp with (red) and without charge reduction (green). Inset structure of P-gp in a DDM micelle.