Kinetics Of Peptide (pHLIP) Insertion And Folding In A Lipid Bilayer Membrane

Abstract

The early stages of folding a membrane protein have been conceptualized in terms of the formation of independently stable transmembrane helices, followed by their association to form a bundle, and then followed by further insertions, rearrangements, and binding events. A part of this notion is the formation of transmembrane helices, which is catalyzed by the translocon for hydrophobic sequences, but which can also occur spontaneously for moderately polar sequences. We study spontaneous insertion and folding across a lipid bilayer of moderately polar membrane peptide pHLIP - pH Low Insertion Peptide. pHLIP has three major states: (I) soluble in water or (II) bound to the surface of a lipid bilayer as an unstructured monomer, and (III) inserted across the bilayer as a monomeric α-helix. The existence of three distinct equilibrium states makes it possible to separate the process of peptide attachment to a lipid bilayer from the process of peptide insertion/folding. The transitions between states could be easily monitored by the changes of tryptophan fluorescence and circular dichroism signals. We performed steady-state and stopped-flow fluorescence and CD measurements to reveal the molecular mechanism of pHLIP insertion and folding within a POPC lipid bilayer and to calculate the activation energy of formation of transmembrane helix. Global mode analysis allowed us to monitor changes of entire tryptophan fluorescence spectrum during the transition from the state II to the state III.