Gas-Phase Protonation Thermodynamics of Biological Lipids: Experiment, Theory, and Implications

Abstract

Phospholipids are important to cellular function and are a vital structural component of plasma and organelle membranes. These membranes isolate the cell from its environment, allow regulation of the internal concentrations of ions and small molecules, and host diverse types of membrane proteins. It remains extremely challenging to identify specific membrane protein-lipid interactions and their relative strengths. Native mass spectrometry, an intrinsically gas-phase method, has recently been demonstrated as a promising tool for identifying endogenous protein-lipid interactions. However, to what extent the identified interactions reflect solution- versus gas-phase binding strengths is not known. Here, the "Extended" Kinetic Method and ab initio computations at three different levels of theory are used to experimentally and theoretically determine intrinsic gas-phase basicities (GB, ΔG for deprotonation of the protonated base) and proton affinities (PA, ΔH for deprotonation of the protonated base) of six lipids representing common phospholipid types. Gas-phase acidities (ΔG and ΔH for deprotonation) of neutral phospholipids are also evaluated computationally and ranked experimentally. Intriguingly, it is found that two of these phospholipids, sphingomyelin and phosphatidylcholine, have the highest GB of any small, monomeric biomolecules measured to date and are more basic than arginine. Phosphatidylethanolamine and phosphatidylserine are found to be similar in GB to basic amino acids lysine and histidine, and phosphatidic acid and phosphatidylglycerol are the least basic of the six lipid types studied, though still more basic than alanine. Kinetic Method experiments and theory show that the gas-phase acidities of these phospholipids are high but less extreme than their GB values, with phosphatidylserine and phosphatidylglycerol being the most acidic. These results indicate that sphingomyelin and phosphatidylcholine lipids can act as charge-reducing agents when dissociated from native membrane protein-lipid complexes in the gas phase and provide a straightforward model to explain the results of several recent native mass spectrometry studies of protein-lipid complexes.

Figure 1:

Chemical structures of the six lipid headgroups studied, shown in their common physiological total charge and protonation states. PhA = phosphatidic acid, PC = phosphatidylcholine, PE = phosphatidylethanolamine, PG = phosphatidylglycerol, PS = phosphatidylserine, SM = sphingomyelin. PhA, PG and PS are shown here in their deprotonated forms, and all other lipids are shown in their net neutral forms. Two diastereomers of PG were evaluated due to a racemic mixture of both isomers being present in natural PG samples from the non-stereoselectivity of the biosynthesis of the glycerol headgroup. R₁ and R₂ represent acyl chains. For the calculations here, methyl groups were used for both R₁ and R₂ to limit computational time, whereas in Extended and Conventional Kinetic Method experiments, R₁ and R₂ were longer acyl chains more typical of physiological membranes (see Methods section).

Figure 2:

Three lowest energy structures of PG 1 in the 1+ charge state found using Monte Carlo conformational searching and optimized at B3LYP/6–311++G**. The global minimum in terms of ΔG/ΔH of formation is conformer a. Conformer b is higher in energy by 0.7/3.8 kJ/mol (ΔG/ΔH, respectively) and conformer c was found to be higher in energy by 1.5/3.5 kJ/mol (ΔG/ΔH, respectively).

Figure 3:

Comparison of experimental Kinetic Method bracketing, literature, and computed GB values for amino acids and model lipids. The arrows for PC and SM brackets indicate that only lower bracket values were found due to the extremely high basicity of these lipids.

Figure 4:

Schematic illustration of lipoprotein Nanodisc CID experiments under moderate activation conditions under which positively charged Nanodiscs lose lipids, but negatively charged Nanodiscs do not. Due to their especially high GB, lipids in positive ion mode are more likely to bear charge, repel other charge sites, and dissociate than are lipids in negative ion mode under similar activation conditions.

Figure 5:

Gas-phase dissociation of a phospholipid-detergent micelle ion with an embedded transmembrane peptide/protein. Extensive activation of the micelle complex may lead to rearrangement of the phospholipids around the peptide/protein to a site that is more energetically favourable in the gas phase and is stabilized by the high GB of the phospholipid headgroup. Possibly even some phospholipids not initially associated directly with the peptide/protein surface in the condensed phase (blue headgroups) may bind to it after activation.