Evolutionary Avoidance of Transmembrane Embedded Arginines is due to Slowed Folding Kinetics

Abstract

Conserved transmembrane (TM) arginines are essential for the transport function of many membrane proteins, such as voltage-gated ion channels. However, among the lipid-facing residues in TM regions, the statistical propensity of arginines is extremely low. The minimal occurrence of TM arginines is often attributed to the thermodynamic cost of inserting an arginine into a hydrophobic lipid environment. But the energetic cost of TM arginine insertion measured by the Moon-Fleming Hydrophobicity scale is only 3 kcal mol-1, implying that Arg should be allowed in the bilayer. To investigate the discrepancy between this moderate thermodynamic cost and the evolutionary avoidance of TM arginines, we utilized Outer Membrane Phospholipase A (OmpLA) as a host for lipid-facing arginine mutations, at three different depths in the bilayer. We probed the effects of the introduction of TM arginines on the secondary structure and enzymatic activity of OmpLA and found that Arg has a minimal impact on the structure and function of OmpLA. We also determined that a TM Arg does not affect the thermodynamic binding of OmpLA to the periplasmic chaperones, SurA and Skp. Molecular dynamics simulations revealed that all TM Args studied form hydrogen bonds with water and phospholipid head groups. Kinetics of folding into large unilamellar vesicles of varying head group compositions reveal that the R-variants exhibit slow folding, even in the presence of the OMP assembly machinery protein, BamA. Slow kinetics are additionally observed for in vivo folding of these R-variants. In addition to a thermodynamic cost for TM arginine insertion our results indicate that a pronounced kinetic barrier may play a role in the evolutionary pressure against TM arginines.