Study of membrane-associated folding and unfolding

The stability and folding of both nonconstitutive and constitutive membrane proteins are strongly constrained by the formation of secondary structures in the lipid bilayer environment driven by the hydrophobic effect and hydrogen bonding. Therefore, the study of the molecular mechanisms of a polypeptide\u27s insertion into a lipid bilayer and the formation of its secondary structure is a key in understanding of the first step of the membrane-associate folding. We have been studying a useful model system for measurement of the thermodynamics and kinetics of peptide insertion and folding across a lipid bilayer. It is based on the pHLIP® peptide (pH Low Insertion Peptide), which has three major states: (I) soluble in water in an unstructured, monomeric state, (II) bound to the surface of a lipid bilayer in an unstructured, monomeric state, and (III) inserted across the bilayer as an ƒÑ-helix. The existence of three distinct equilibrium states makes it possible to separate the process of the peptide\u27s attachment to a lipid bilayer from the process of the peptide\u27s insertion/folding. Previously we showed that pHLIP insertion is associated with the protonation of Asp residues, which leads to an increase of the pHLIP hydrophobicity that triggers the folding and insertion of the peptide across a lipid bilayer. Thus, the insertion/folding and exit/unfolding of the pHLIP peptides can be triggered by pH jumps. The main goal of this study is to elucidate mechanism of membrane-associate folding/unfolding. The steady-state fluorescence, circular dichroism (CD) and oriented CD, as well as stopped-flow fluorescence and CD investigation was carried out with several pHLIP variants. We showed that the pHLIP insertion occurs in several steps, with rapid (0.1 s) interfacial helix formation followed by a much slower (45 s) insertion pathway to give a transmembrane helix. The reverse process of unfolding and the peptide’s exit from the bilayer core, which can be induced by a rapid rise of the pH from acidic to basic, proceeds ∼800 times faster than the folding/insertion and through different intermediate states. To address the question why it takes 1000 times longer for the helix to insert into a bilayer after it is formed on the membrane surface, and to elucidate the nature of the intermediates along the folding and unfolding pathways we investigated several pHLIP variants with different number of charged residues at the membrane-inserting end, and three single-Trp variants, where the Trp residue was placed at the beginning, middle and end of the transmembrane helix. We confirmed that all pHLIP variants preserve pH-dependent properties of interaction with a membrane. We found that the number of protonatable residues at the inserting end does not affect the formation of helical structure, but correlates with the time of the peptide’s insertion into a membrane, the number of the intermediate states on the folding pathway, and the rates of the peptide’s unfolding and exit. We concluded that the existence of intermediate states on the folding and unfolding pathways are nonmandatory and, in the simple case of a polypeptide with a non-charged and non-polar inserting end, the folding and unfolding are all-or-none transitions. The model of membrane-associated insertion/folding and exit/unfolding is proposed. The obtained results provide insight in the mechanism of membrane protein folding and are very valuable for the development of new approaches in targeting of acidic diseased tissue and translocation of polar cargo molecules across cellular membranes