Mechanism of Spontaneous Insertion/Folding of Peptides in Membrane

The folding of helical membrane proteins can be conceptualized in terms of distinct steps:

i) peptide insertion and folding to form independently stable helices across a lipid bilayer

ii) helix association to form a helix bundle intermediate

iii) further rearrangements of protein structure and/or binding of prosthetic groups to achieve a functional state.

Insertion of most membrane proteins is facilitated in vivo by complex molecular machines, such as the translocon, which assists in placing hydrophobic sequences across the bilayer. In contrast to more hydrophobic sequences, moderately polar transmembrane domains of C terminally anchored proteins can postranslationally translocate themselves into membranes in a translocon-defective yeast strain.

We are currently 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

(III) inserted across the bilayer as an α-helix (see Figure).

At neutral pH in water, pHLIP carries several negative charges in the form of Asp and Glu residues. Low pH induces protonation of Asp residues, which leads to an increase in pHLIP hydrophobicity that immediately (within seconds) biases the equilibrium among the states and causes insertion of the peptide into a lipid bilayer membrane. 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. pHLIP is a unique example of a water-soluble, membrane inserting peptide, in that it exists in monomeric form in all three major states.

This project is performed in collaboration with the Laboratory of Prof. Donald Engelman, Yale University.

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A schematic representation of pHLIP interaction with the lipid bilayer of a membrane is shown. a) State I correspond to the peptide in solution at normal and basic pHs. By addition of vesicles, the unstructured peptide is adsorbed on the membrane surface. State II, type I interactions, initial steps in peptide adsorption to a membrane, occur at a low lipid:peptide ratio, when the total accessible membrane surface is not enough to accommodate the full length of peptide on it. We reason that the hydrophobic motif near the C-terminus is adsorbed first (red sequence). By addition of more vesicles, the transition to the type II (state II) interaction is seen, at a high lipid:peptide ratio when there is enough accessible space on a membrane for peptides to freely occupy lipid surface area without competing with each other. A drop of pH leads to the protonation of Asp residues, increasing peptide hydrophobicity, and resulting in the insertion and formation of a transmembrane alpha-helix. Our thermodynamic measurements suggest the existence of 3 major populations of lipids: i) lipids interacting with the peptide directly (lipids with blue head groups); ii) lipids, not interacting with the peptide directly, but influenced by the interaction (lipids with cyan head groups,possibly in both leaflets) and iii) lipids that are not involved in the interaction with pHLIP (lipids with yellow head groups). The values for energy are taken for 37°C. b) The pHLIP sequence is presented, with the presumed transmembrane part in bold (based on the helix seen in bacteriorhodopsin). Red is used to denote the hydrophobic sequence and the C-terminus, which are expected to initially interact with the bilayer. (Reshetnyak et al., 2008, PNAS).

We show that insertion occurs in several steps, with rapid (0.1 sec) interfacial helix formation followed by a much slower (100 sec) insertion pathway to give a transmembrane helix. The reverse process of unfolding and peptide exit from the bilayer core, which can be induced by a rapid rise of the pH from acidic to basic, proceeds  400 times faster than folding/insertion and through different intermediate states. In the exit pathway, the helix-coil transition is initiated while the polypeptide is still inside the membrane. The peptide starts to exit when about 30% of the helix is unfolded, or the polypeptide exits lipid bilayer immediately after unfolding, which leads to pinch of the membrane around shorten helical part of the peptide.

Membrane-associated pHLIP peptide folding and insertion across a POPC bilayer (a-c) and unfolding and exit (d-f) from the core of the bilayer were monitored by stopped-flow CD and fluorescence. Polypeptide folding and unfolding were induced by the rapid mixing of pHLIP-POPC solutions with diluted HCl or NaOH to give pH4 or pH8, respectively. The changes of intensity of CD (a, d) and fluorescence (b, e) were recorded at 225 nm (CD) and through a 320 nm cutoff filter using an excitation wavelength of 280 nm (fluorescence) at 25°C. The fluorescence signal recorded over 80 sec was corrected for photobleaching. The CD and fluorescence data were fitted by kinetic models with one or three intermediate states by using equations (3 and 4 supplementary information) (the fitting curves are red). In the case of the helix-coil transition (d) the experimental noise did not allow to reveal statistically significant differences between solutions with no (blue line) or a single intermediate (red line), thus both fitting curves and calculated parameters are shown. The changes in the entire fluorescence spectrum during folding (c) and unfolding (f) were recorded in a global mode with the emission monochromator at the excitation wavelength of 275 nm to minimize the contribution of the scattered light component at short wavelengths (spectra were corrected for the instrument sensitivity) (Andreev et al, 2010, PNAS).

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