Like Fish Out of Water: Membrane Proteins in Detergent Micelles

Abstract

Biophysical studies of structure-function relations of membrane proteins are often done in detergent micelles. However, the scarce molecular understanding of the way in which such micelles form and organize around guest proteins makes it difficult to evaluate the relation between the experimental models and the properties of the membrane proteins in their native environment. Because experimental conditions were shown to have a major effect on structure-function relations of the leucine transporter (LeuT), a prokaryotic homolog of the mammalian neurotransmitter:sodium symporter proteins and a prototype for their study, we investigated LeuT in dodecyl-β-maltoside (DDM) detergent micelles. Atomistic molecular dynamics (MD) simulations revealed the formation of a constant-sized “detergent core” within 4A of LeuT consisting of ∼120 DDM molecules regardless of the DDM-to-protein number ratio. However, we found that the aggregation number of the protein-detergent complex (i.e., the number of DDMs associated with the micelle surrounding the protein) depends on detergent concentration. Notably, this aggregation number appears to determine the extent of detergent penetration into the LeuT extracellular vestibule. Thus, we observed DDM penetration into LeuT via two pathways, but the penetration was detected only in the constructs with high detergent content. In constructs with low DDM concentration, any detergent penetration was at most transient. Entering from the “side” of LeuT, a DDM molecule passes between the extracellular segments of transmembrane helix 6 (TMH6) and TMH10, and interacts with TMH1 residues Arg30 and Gln34 in the secondary substrate-binding site (S2) of LeuT. When insertion is “from the top”, the DDM passes by extracellular loop 4 (ECL4) before it penetrates the S2 site at the level of Phe320 (in ECL4) and Leu400 (in TMH10). These findings are discussed in light of experiments that established a modulatory effect of DDM concentration on LeuT activity.