The Role of Annular Lipids in the Mechanism of Membrane Protein Solubilization and Formation of Functionally Competent Proteomicelles

Abstract

The “solubilization” procedure in which membrane proteins are extracted from their native lipid membranes and transferred into detergent micelles (proteomicelles) is a critical step in the purification of the protein for subsequent structure/function studies. In some notable examples it has become clear, however, that the properties of the proteins in proteomicelles depend on the detergent type and its concentration relative to the residual membrane lipids originating from the preparation. To reveal the molecular mechanism of the solubilization process and the effect of various lipid-to-detergent (L-D) ratios on the properties of the system, we performed extensive atomistic molecular dynamics simulations. We previously explored the properties of micelles formed by dodecyl-β-maltoside (DDM) detergent around the leucine transporter (LeuT), a bacterial homolog of the neurotransmitter:sodium symporter family, and showed how the DDM concentration determines the extent of detergent penetration into the functionally relevant secondary substrate (S2) binding site in LeuT. Here we present computational studies of LeuT embedded in proteomicelles at different L-D ratios exploring configurations of LeuT proteomicelles at different stages of the protein solubilization process. Our results reveal that at a sparse lipid annulus around the transporter, consisting of no more than ∼10 lipids, DDM partitions into the LeuT, engaging with the residues in the S2 site. Higher lipid concentrations protect the S2 site from DDM penetration. In contrast to DDM, MNG-3 (lauryl maltose-neopentyl glycol) detergent, an amphiphile known to better stabilize membrane proteins, does not penetrate the transporter even in the absence of lipids. Parallel experimental measurements of ligand binding to LeuT in the presence of increasing MNG-3 concentrations revealed that the functionality of the S2 site was fully preserved even at the highest (0.5%) MNG-3 concentration probed.