Biophysics of the membrane interface and its involvement in protein targeting and translocation

A. Watts and T.J.T. Pinheiro

Department of Biochemistry, South Parks Road, University of Oxford, Oxford OX| 3QU, U.K.

Introduction

The initial site of association for any component that may partition into and then, as one possibility, traverse a membrane is the polar/apolar interface of the membrane. Whether or not a protein or lipid acts as the target site, such associations are driven initially, and possibly subsequently, by electrostatic forces. These forces are important not only in ionic interactions and conductance effects, but also in determining the structure and activity of membrane proteins, including protein insertion and translocation. Here, the relevant thermodynamic and electrostatic aspects of membrane protein association and insertion will be reviewed. As specific examples of such associations, the mode of interaction and kinetics of association of several peptides and proteins will be presented using a range of biophysical approaches, although it should be stated that the area is highly complex, and no simple explanations exist for any systems, and much information is piecemeal and incomplete. This short resume cannot hope to include every aspect of the topic, but some indication of contemporary methods and results will be given.

Thermodynamic and practical consideration of protein–membrane associations

When considering membrane protein insertion and translocation, the thermodynamics of the interactions are important, driven, as they are initially, by electrical forces. The free energy of peptide binding to a membrane can be approximated from:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]

where ΔGel denotes the electrostatic effects (see below), ΔGimm is the positive binding energy caused by peptide immobilization, ΔGfob is the energy gained from the hydrophobic effects, ΔGpol is the energy contribution from backbone and side-chain hydrogen bonding, and ΔGlip describes the lipid perturbation effects. Any configurational energy associated with peptide-membrane binding is included within the ΔGpol term.

Biophysical methods are able to allow some estimates of these various terms, although too little is known at present to permit complete descriptions of the association mechanisms. The value of ΔGimm for the peptide on the membrane surface, when compared with isotropically moving peptide, is not really known. However, when compared with the more favourable disordered unfolded helixforming peptide, the folded and thus more ordered helix has an unfavourable energy of ΔGimm ~5.23 kj · mol-1 (~1.25 kcal · mol-1) per peptide bond, giving approx. 84–126 kj · mol-1 (20-30 kcal · mol-1) for a 20-amino-acid helix. Against this, a similar transmembrane helix would form 16 hydrogen bonds in the non-aqueous environment of the bilayer core, giving a ΔGpol of approx.—402 kj · mol-1 (-96 kcal · mol-1), with only small contributions from van der Waals interactions between lipids and side chains. Estimates of ΔGimm + ΔGfob of at least 84 kj · mol-1 (20 kcal · mol-1) have been made for the desolvation/hydration of signal peptides. However, it has been pointed out that the 'macroscopic' and 'microscopic' hydrophobic effects in membranes and protein binding sites are very different in magnitude and that highly curved surfaces can produce an anomalous, high, hydrophobic energy of binding. Furthermore, for peptide insertion and folding, and again on energetic grounds, the insertion of an unfolded chain is extremely unfavourable [ΔGimm +176 kj · mol-1 (+42 kcal · mol-1)] when compared with insertion of a folded chain [ΔGimm -126 kj · mol-1 (-30 kcal · mol-1)] (20–22 residues). From similar arguments, assembled ß-structures are also unfavourably inserted into a bilayer. Based on such considerations, it has been argued that a polypeptide coil cannot be inserted into a bilayer and then fold, but rather a secondary structure must be formed either in the aqueous phase or, at the latest, at the membrane/water interface before insertion.

The maximum energy from inserting the hydrophobic residues of the LamB wild-type peptide into the bilayer (ΔGfob) is approx. -381 kj · mol-1 (-91 kcal · mol-1), which is very substantial and favourable. Also, the free energy required for transfer of unbonded polar groups is much higher [OH, +16.7 kj · mol-1 (+4.0 kcal · mol-1); -NH2, +20.9 kj · mol-1 (+ 5.0 kcal · mol-1); COOH, +20.1 kj · mol-1 (+4.8 kcal · mol-1); C = O, +8.4 kj · mol-1 (+2.0 kcal · mol-1)] than for bonded -NHO = C pairs [+2.3 kj · mol-1 (+0.55 kcal · mol-1)].

Lipid perturbation effects play an insignificant role in the energetics of peptide insertion and translocation. Ordering of lipids around the peptide may be significant, but this is offset by the favourable interaction of acyl chains with hydrophobic peptide residues. Partially embedded peptides, or those on the bilayer surface, cannot be assessed so readily and lipid phase separation, cavity formation and lipid head-group perturbations are all relatively poorly described. However, as a general contribution to the entropic changes upon peptide binding, the restriction of a peptide on the bilayer surface,...