Stephen H. White

Recognition of transmembrane helices by the endoplasmic reticulum translocon

Membrane proteins depend on complex translocation machineries for insertion into target membranes. Although it has long been known that an abundance of nonpolar residues in transmembrane helices is the principal criterion for membrane insertion, the specific sequence-coding for transmembrane helices has not been identified. By challenging the endoplasmic reticulum Sec61 translocon with an extensive set of designed polypeptide segments, we have determined the basic features of this code, including a ‘biological’ hydrophobicity scale. We find that membrane insertion depends strongly on the position of polar residues within transmembrane segments, adding a new dimension to the problem of predicting transmembrane helices from amino acid sequences. Our results indicate that direct protein–lipid interactions are critical during translocon-mediated membrane insertion.

Molecular code for transmembrane-helix recognition by the Sec61 translocon

Transmembrane α-helices in integral membrane proteins are recognized co-translationally and inserted into the membrane of the endoplasmic reticulum by the Sec61 translocon. A full quantitative description of this phenomenon, linking amino acid sequence to membrane insertion efficiency, is still lacking. Here, using in vitro translation of a model protein in the presence of dog pancreas rough microsomes to analyse a large number of systematically designed hydrophobic segments, we present a quantitative analysis of the position-dependent contribution of all 20 amino acids to membrane insertion efficiency, as well as of the effects of transmembrane segment length and flanking amino acids. The emerging picture of translocon-mediated transmembrane helix assembly is simple, with the critical sequence characteristics mirroring the physical properties of the lipid bilayer.

HYDROPHOBIC INTERACTIONS OF PEPTIDES WITH MEMBRANE INTERFACES

The thermodynamic principles underlying the structural stability of membrane proteins are difficult to obtain directly from whole proteins because of intractable problems related to insolubility in the aqueous phase and extreme stability in the membrane phase. The principles must therefore be surmised from studies of the interactions of small peptides with lipid bilayers. This review is concerned with the hydrophobic interactions of such peptides with the interfacial regions of lipid bilayers. We first develop a general framework for thinking about the thermodynamics of membrane protein stability that centers on interfacial interactions and review the structural and chemical evidence that supports this interface-centered point of view. We then describe an experimentally determined whole-residue interfacial hydrophobicity scale that reveals the central role of the peptide bond in partitioning and folding. Finally, we consider the complexity and diversity of interfacial interactions revealed by differences between side-chain hydrophobicities determined using different classes of peptides.

Structure, function, and membrane integration of defensins

Defensins comprise a structural class of small cationic peptides that exert broad-spectrum antimicrobial activities through membrane permeabilization. Their predominantly beta-sheet structure, stabilized by three disulfide bonds, distinguishes them from other antimicrobial peptides which typically form amphiphilic helices. Defensins bind to membranes electrostatically and subsequently form apparently multimeric pores. Recent structural and biophysical studies are beginning to provide insights into the process of permeabilization.

Biophysical dissection of membrane proteins

The first atomic-resolution structure of a membrane protein was solved in 1985. Twenty-four years and more than 180 unique structures later, what have we have learned? An examination of the atomic details of several diverse membrane proteins reveals some remarkable biophysical features and suggests that we can expect to achieve much more in the decades to come.

Structure, Location, and Lipid Perturbations of Melittin at the Membrane Interface

Melittin is arguably the most widely studied amphipathic, membrane-lytic alpha-helical peptide. Although several lines of evidence suggest an interfacial membrane location at low concentrations, melittins exact position and depth of penetration into the hydrocarbon core are unknown. Furthermore, the structural basis for its lytic action remains largely a matter of conjecture. Using a novel x-ray absolute-scale refinement method, we have now determined the location, orientation, and likely conformation of monomeric melittin in oriented phosphocholine lipid multilayers. Its helical axis is aligned parallel to the bilayer plane at the depth of the glycerol groups, but its average conformation differs from the crystallographic structure. As observed earlier for another amphipathic alpha-helical peptide, the lipid perturbations induced by melittin are remarkably modest. Small bilayer perturbations thus appear to be a general feature of amphipathic helices at low concentrations. In contrast, a dimeric form of melittin causes larger structural perturbations under otherwise identical conditions. These results provide direct structural evidence that self-association of amphipathic helices may be the crucial initial step toward membrane lysis.

Protein folding in membranes: determining energetics of peptide-bilayer interactions.

Although the problem of the folding of soluble proteins continues to resist solution, we at least have a strong understanding of the general thermodynamic principles1,2 and have available a wealth of thermodynamic data.3-5 The study of membrane protein folding and stability is much less advanced: Some general principles are emerging,6-9 but the amount of thermodynamic data available remains quite limited. The energetics of the partitioning of peptides into membranes constitutes one especially important class of data. We will demonstrate how such data can be used for clarifying the folding of peptides and small proteins in membranes and then describe the principles and methods used for determining the energetics of the partitioning of peptides into bilayer membranes.

How Translocons Select Transmembrane Helices

Like all cellular proteins, membrane proteins are synthesized by ribosomes. But unlike their soluble counterparts, highly hydrophobic membrane proteins require auxiliary machineries to prevent aggregation in aqueous cellular compartments. The principal machine is the translocon, which works in concert with ribosomes to manage the orderly insertion of alpha-helical membrane proteins directly into the endoplasmic reticulum membrane of eukaryotes or into the plasma membrane of bacteria. In the course of insertion, membrane proteins come into thermodynamic equilibrium with the lipid membrane, where physicochemical interactions determine the final three-dimensional structure. Much progress has been made during the past several years toward understanding the physical chemistry of membrane protein stability, the structure of the translocon machine, and the mechanisms by which the translocon selects and inserts transmembrane helices. We review this progress and consider the connection between the physical principles of membrane protein stability and translocon selection of transmembrane helices.

MPEx: A tool for exploring membrane proteins

Hydropathy plot methods form a cornerstone of membrane protein research, especially in the early stages of biochemical and structural characterization. Membrane Protein Explorer (MPEx), described in this article, is a refined and versatile hydropathy‐plot software tool for analyzing membrane protein sequences. MPEx is highly interactive and facilitates the characterization and identification of favorable protein transmembrane regions using experiment‐based physical and biological hydrophobicity scales. Besides allowing the consequences of sequence mutations to be examined, it provides tools for aiding the design of membrane‐active peptides. MPEx is freely available as a Java Web Start application from our web site at http://blanco.biomol.uci.edu/mpex.

Sizing membrane pores in lipid vesicles by leakage of co-encapsulated markers: pore formation by melittin.

Many toxins and antimicrobial peptides permeabilize membrane vesicles by forming multimeric pores. Determination of the size of such pores is an important first step for understanding their structure and the mechanism of their self-assembly. We report a simple method for sizing pores in vesicles based on the differential release of co-encapsulated fluorescently labeled dextran markers of two different sizes. The method was tested using the bee venom peptide melittin, which was found to form pores of 25-30 A diameter in palmitoyloleoylphosphatidylcholine (POPC) vesicles at a lipid-to-peptide ratio of 50. This result is consistent with observations on melittin pore formation in erythrocytes (Katsu, T., C. Ninomiya, M. Kuroko, H. Kobayashi, T. Hirota, and Y. Fujita 1988. Action mechanism of amphipathic peptides gramicidin S and melittin on erythrocyte membrane Biochim. Biophys. Acta. 939:57-63).