Reorganization of lipid domain structure in membranes by a transmembrane peptide: an ESR spin label study on the effect of the Escherichia coli outer membrane protein A signal peptide on the fluid lipid domain connectivity in binary mixtures of dimyristoyl phosphatidylcholine and distearoyl phosphatidylcholine

The effect of a transmembrane peptide on the domain structure of a two-component, two-phase lipid bilayer composed of dimyristoyl phosphatidylcholine (DMPC) and distearoyl phosphatidylcholine (DSPC) was examined by spin label electron spin resonance (ESR) spectroscopy. The peptide, pOmpA, is the hydrophobic, 25-residue signal sequence of the outer membrane protein A from Escherichia coli. Nitroxide derivatives of the phospholipid DSPC, 16-DSPCSL, and of the pOmpA signal peptide, pOmpA-IASL, were used as probes. The first-derivative lineshapes of the ESR spectra were analyzed using a normalized intensity ratio, R, that gives information on the average sizes of the disconnected fluid domains and their point of connectivity (Sankaram, M.B., D. Marsh, and T.E. Thompson. 1992. Biophys. J. 63:340–349). In the absence of the peptide, the number of fluid lipid domains does not vary with the fraction of lipid that is in the fluid phase, and phase conversion is accomplished solely by changes in the domain size. The phase boundaries of the lipid mixture remain largely unchanged by the presence of the peptide at mole fractions up to 0.02, but both the size and number of the fluid domains is changed, and the point at which they become connected is shifted to lower fractions of the fluid phase. In addition, the number of domains in the presence of the peptide no longer remains constant but increases from a domain density at low fractions of the fluid phase that is much lower than that in the absence of peptide to one that is comparable to the natural state in the absence of peptide at the point of domain connectivity. A simple model is presented for the process of domain fission, where the latter is determined by a balance between the effects of peptide concentration in the fluid domains, the line tension at the domain boundaries, and the distributional entropy of the domains

Chaperones and protein folding

The folding and translocation of many newly synthesized proteins in the cell is kinetically assisted by ubiquitous, abundant, specialized proteins known as molecular chaperones. These components generally recognize hydrophobic surfaces, exposed specifically by non-native conformations, through their own solvent-exposed hydrophobic surfaces, with different classes of chaperone recognizing such surfaces in the context of extended (Hsp70) vs. collapsed (Hsp60/chaperonin) topology of substrate protein. Such binding prevents substrate proteins from misfolding and from forming multimolecular aggregates. Chaperone-bound proteins are then released from Hsp70 and Hsp60 machines via the binding of ATP to chaperone domains physically separated from the substrate protein binding domains, via allosterically directed conformational changes. Molecular chaperones also act under stress conditions, where polypeptide chains are subject to misfolding, preventing aggregation and restoring the native state. The small heat shock proteins (sHsps) are oligomeric assemblies that participate with the other chaperones in binding non-native states under such conditions. Finally, Hsp90 is an abundant clamp-shaped chaperone that participates in binding and maturation of a variety of substrate proteins via an ATP-directed cycle. This chapter reviews the structure and mechanism of action of these chaperones, with special attention directed to the variety of biophysical methods employed to reaching our current understanding