First step in folding of nonconstitutive membrane proteins: spontaneous insertion of a polypeptide into a lipid bilayer and formation of helical structure

There are two questions we would like to address: 1) what is the molecular mechanism of a polypeptide insertion into a lipid bilayer and formation of transmembrane helix? 2) Are there any transient changes of a lipid bilayer in process of a polypeptide insertion and folding? As a convenient system we are studying pHLIP (pH (Low) Insertion Peptide) insertion into a membrane and folding, which is modulated by pH. Previously we showed, that insertion of pHLIP occurs in several steps, with rapid (0.1 sec) interfacial helix formation followed by a much slower (100 sec) insertion pathway to form a transmembrane helix. The reverse process of unfolding and peptide exit from the bilayer core, which can be induced by a rapid pH jump from acidic to basic, proceeds much faster than folding/insertion and through different intermediate states. We designed two truncated pHLIP-variants by removing Asp and Glu residues from the C-terminus, which goes across a membrane. Our kinetic studies indicate that truncated versions of pHLIP insert across a membrane much faster than original (WT) sequence and with less number of intermediate states. Thus, the rate of insertion directly depends on probability of protonation of C-terminal Asp and Glu residues, and translocation of the C-terminus across a bilayer. To understand what are the structural intermediates along the folding pathway, we designed three single-tryptophan pHLIP-variants, where a Trp residue is positioned at the beginning, middle and end of the transmembrane part of the peptide. To study changes, which might occur with a lipid bilayer in a process of peptide insertion and folding, we employed SAXS (small-angle x-ray scattering) technique. A new kinetic model of pHLIP insertion/exit will be discussed.