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The structures of integral membrane proteins are of interest to the field of structural biology, but they have been less than amenable for determination by x-ray crystallography. Crystals of at least 20 membrane proteins have been obtained (1), but in only a few cases have the crystals been of suitable quality to permit the resolution of atomic structure (2). With the hypothesis that small-molecule detergents, as required to solubilize membrane proteins, are in some way responsible for the disorder within the crystals (3), we attempted to design homogeneous peptides as detergents, "peptitergents" that would lead to a more homogeneous, well-ordered complex for crystallography. Peptitergents are amphipathic peptides designed to sequester the hydrophobic membrane-spanning region of membrane proteins by packing around the protein in a rigid, well-ordered, parallel alpha-helical arrangement. The first peptitergent, PD sub 1 , was designed, synthesized, and crystallized by itself and found to form an antiparallel four-helix bundle, a structure that is of interest from the point of view of de novo protein design. It also interacted with the integral membrane proteins bacteriorhodopsin and rhodopsin to maintain the majority of protein in solution for several days (Fig. 1). (Figure 1 omitted) In contrast, PD sub 1 did not maintain PhoE porin solubility.
The peptide was designed as a 24-residue amphipathic alpha helix (Fig. 2) with a hydrophobic surface 30 (Character omitted) in length, long enough to traverse the membrane-spanning region of an integral membrane protein. (Figure 2 omitted) The hydrophobic surface was designed with alanines projecting from the center and leucines from the sides to form a wide, flat face that would permit interaction with the various surfaces of a transmembrane protein. The leucines were placed to allow them to interact with leucines on other PD sub 1 helices through hydrophobic contacts. Glutamic acid and lysine were used in an attempt to form salt bridges at the helix termini that would stabilize the helix termini; the salt bridges were designed to be at positions i and i + 4 and to be oriented so as to stabilize the helix dipole (4). The helix termini were capped by acetylation of the amino terminus and conversion of the carboxy terminus to an amide to neutralize the potentially destabilizing terminal charges that would interact unfavorably...