From The Cover: De novo design of defined helical bundles in membrane environments

From The Cover: De novo design of defined helical bundles in membrane environments
approved September 14, 2004 (received for review May 11, 2004)
Published online before print October 14, 2004
Baar Bilgi?er *, and Krishna Kumar *, ,
*Department of Chemistry, Tufts University, Medford, MA 02155; and Cancer Center, Tufts?New England Medical Center, Boston, MA 02110
Edited by William F. DeGrado, University of Pennsylvania, Philadelphia, PA
Control of structure and function in membrane proteins remains a formidable challenge. We report here a new design paradigm for the self-assembly of protein components in the context of nonpolar environments of biological membranes. An incrementally staged assembly process relying on the unique properties of fluorinated amino acids was used to drive transmembrane helix?helix interactions. In the first step, hydrophobic peptides partitioned into micellar lipids. Subsequent phase separation of simultaneously hydrophobic and lipophobic fluorinated helical surfaces fueled spontaneous self-assembly of higher order oligomers. The creation of these ordered transmembrane protein ensembles is supported by gel electrophoresis, circular dichroism spectroscopy, equilibrium analytical ultracentrifugation, and fluorescence resonance energy transfer.
The functional properties of proteins are intimately linked to their shape in solution. Proteins spontaneously fold into exquisite three-dimensional structures, representing a delicate balance between protein?protein and protein?solvent interactions. Water-soluble proteins achieve an energetically favorable equilibrium by sequestering nonpolar residues in the interior and placing polar and charged groups on the surface. This simple idea has been exploited as a generalized design paradigm for constructing water-soluble protein architectures (1?4). Indeed, binary patterning with polar and nonpolar amino acids has been successfully used to direct the folding of such proteins (5, 6). Rees et al. (7) have quantitatively analyzed the hydrophobicities of interior versus membrane exposed residues for putative transmembrane helical sequences. They concluded that membrane proteins exhibit a far less pronounced asymmetry in the distribution of polar and nonpolar side chains. In other words, the hydrophobic effect as an organizing force is absent in the long acyl chain region of bilayers. The design of selective protein?protein interfaces in the context of biological membranes is therefore a considerable challenge and remains an unsolved problem in structural biology (8?13).
Naturally occurring protein?protein interfaces either contain elements of polar specificity, for example hydrogen bonding or salt bridges, or are composed of complementary hydrophobic patches containing side chains that maximize van der Waals interactions. DeGrado, Engelman, and colleagues (14?17) have elegantly demonstrated the homomeric association of transmembrane helices by introduction of residues containing polar side chains, including those of Asn, Gln, Asp or Glu (14?17), that can participate in interchain hydrogen bonding. This strategy provides appreciable driving force for oligomerization and has recently been implicated in disease states where a neutral to charged (V232D) mutation within the membrane results in loss of function due to altered assembly and alignment of the cystic fibrosis transmembrane conductance regulator (18). This finding suggests that, in the presence of a number of potential hydrogen-bonding partners in the biological milieu, specificity using this strategy may be difficult to achieve. On the other hand, for proteins embedded within membrane bilayers, tuning the differential van der Waals affinity of protein side chains for one another in the midst of a sea of lipid hydrocarbon tails also proves difficult. What is required from a protein design perspective is an orthogonal hydrophobe that partitions into nonpolar environments away from water but subsequently phase separates from the hydrocarbon lipids.
Highly fluorinated compounds have long been known to have a low propensity to interact with other materials (19). Recent studies have used amino acid side chains incorporating this type of chemical functionality to fashion a new type of protein?protein interaction motif that is simultaneously hydrophobic and lipophobic (20?25). The selectivity in these interfaces is derived from the extra-biological and remarkable properties of highly fluorinated side chains (21). These structures provide an avenue to selectively oligomerize membrane-soluble protein components. We envisioned a two-step assembly for the folding and oligomerization of transmembrane protein segments. First, hydrophobic peptides would partition into micelles. Second, due to phase separation properties of appropriately placed fluorinated amino acids, the peptides would self-assemble within the lipid environment into predetermined structures (Fig. 1). Here, we show that fluorinated surfaces are remarkably effective in mediating helix?helix interactions in transmembrane protein domains.
Fig. 1. Schematic diagram depicting the two-step self-assembly of membrane-soluble protein segments in micelles. The designed peptides are extremely hydrophobic, are only minimally soluble in water, and partition readily into micelles forming -helices. One face of the helix exposes a string of hexafluoroleucine residues, shown in space-filling representation that promotes the formation of higher order aggregates. Gray, C; red, O; blue, N; green, F; purple, backbone. Only the backbone (depicted as a helix) and the core packing residues are shown for clarity.

By incorporation of fluorinated side chains, which render selected helical protein surfaces simultaneously hydrophobic and lipophobic, we introduce here a binary patterning scheme suitable for use in biological membranes. We demonstrate the folding and oligomerization of several 29-residue polypeptides in micellar detergents using this strategy. The results described here pave the way for the design of increasingly complex and sophisticated membrane protein architectures involving multiple protein components.
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