Step toward Probing the Nonannular Belt of Membrane Proteins

Abstract

Integral membrane proteins are embedded in the biological membrane, where they carry out numerous biological processes. Although lipids present in the membrane are crucial for membrane protein function, it remains difficult to characterize many lipid binding events to membrane proteins, such as those that form the annular belt. Here, we use native mass spectrometry along with the charge-reducing properties of trimethylamine N-oxide (TMAO) to characterize a large number of lipid binding events to the bacterial ammonia channel (AmtB). In the absence of TMAO, significant peak overlap between neighboring charge states is observed, resulting in erroneous abundances for different molecular species. With the addition of TMAO, the weighted average charge state (Z avg) was decreased. In addition, the increased spacing between nearby charge states enabled a higher number of lipid binding species to be observed while minimizing mass spectral peak overlap. These conditions helped us to determine the equilibrium binding constants (K d) for up to 16 lipid binding events. The binding constants for the first few lipid binding events display the highest affinity, whereas the binding constants for higher lipid binding events converge to a similar value. These findings suggest a transition from nonannular to annular lipid binding to AmtB.

Emergence of mass spectrometry detergents for membrane proteomics

“…Third, charge reduction can increase the difference in the mass-to-charge ratio ( m/z ) between signals of membrane protein apo form, ligand-bound states, and post-translational modifications. The latter aspect can be important for the analysis of individual protein–ligand complexes or proteoforms whose m/z channels would overlap in the case of highly charged membrane protein ions [ 80 ]. Methods for membrane protein charge reduction include (i) increasing the basicity of the detergents’ functional groups to increase the affinity for capturing charge, for example, through the implementation of triazole, amine-oxide, or cis / trans azobenzene [ 58 , 60 , 68 , 77 ], (ii) implementing charge-chelating groups into the detergent head group, such as in the case of C8E4 or OGDs, to increase the affinity for capturing charge [ 60 , 77 ], (iii) treating the ESI plume with acetonitrile vapor [ 79 ], (iv) adding solution additives, such as imidazole and its derivatives [ 79 , 81 ], amine oxides [ 82 – 84 ], amines [ 85 , 86 ], and alkali metal acetate salts [ 87 ], and (v) detergent exchange from non-charge-reducing detergent into a charge-reducing detergent [ 60 ].…”

Native mass spectrometry of membrane protein-lipid interactions in different detergent environments

“…For samples without charge-reducing reagents, the same volume of aqueous ammonium acetate was used instead of charge-reducing reagents as previously described. 19 Native Mass Spectrometry (MS). Samples were loaded into a gold-coated borosilicate nanoelectrospray ionization emitters prepared in-house 13 at room temperature and introduced into an Exactive Plus EMR Orbitrap Mass Spectrometer (Thermo Scientific).…”

“…17 Additionally, More importantly, charge reduction increases the peak spacing, reducing the likelihood of mass spectral peak overlap and improving resolution for higher-order ligand-bound states. 18,19 Various native MS approaches have been developed to generate charge-reduced ions using nanoelectrospray ionization (nanoESI). [20][21][22] One strategy involves the addition of chargereducing molecules.…”

Native mass spectrometry of proteoliposomes containing integral and peripheral membrane proteins

“…AmtB from Escherichia coli (UniProt P69681) were expressed and purified as previously descried. 65 In brief, E. coli AmtB was expressed in E. coli C43 (DE3) (Lucigen) as a N -terminal HRV3C protease cleavable 10x His-tag and maltose binding protein (MBP) in TB (IBI Scientific). The cells were induced with 0.5 mM IPTG at an OD 600 reached between 0.6–0.8 and expressed overnight at 20 °C while shaking.…”