Understanding the mechanisms of action of membrane proteins requires the elucidation of their structures to high resolution. The critical step in accomplishing this by x-ray crystallography is the routine availability of well-ordered three-dimensional crystals. We have devised a novel, rational approach to meet this goal using quasisolid lipidic cubic phases. This membrane system, consisting of lipid, water, and protein in appropriate proportions, forms a structured, transparent, and complex three-dimensional lipidic array, which is pervaded by an intercommunicating aqueous channel system. Such matrices provide nucleation sites (''seeding'') and support growth by lateral diffusion of protein molecules in the membrane (''feeding''). Bacteriorhodopsin crystals were obtained from bicontinuous cubic phases, but not from micellar systems, implying a critical role of the continuity of the diffusion space (the bilayer) on crystal growth. Hexagonal bacteriorhodopsin crystals diffracted to 3.7 Å resolution, with a space group P6 3 , and unit cell dimensions of a ‫؍‬ b ‫؍‬ 62 Å, c ‫؍‬ 108 Å; ␣ ‫؍‬ ␤ ‫؍‬ 90؇ and ␥ ‫؍‬ 120؇.

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Hydrophobic interactions with the greasy slide guide the sugar into and through the channel constriction. The glucosyl-binding subsites at the channel constriction confer stereospecificity to the channel along with the ability to scavenge substrate at low concentrations.

Our comparison reveals that the overall structure of OmpF is not influenced by crystal lattice constraints and, thus, presumably bears close resemblance to the in vivo structure. The tetragonal crystal structure has provided the starting model for the phasing of neutron diffraction data obtained from this crystal form, as described in an accompanying article.

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Porin spans the outer membrane of Escheichia coli with most of the protein embedded within the membrane. It lacks pronounced hydrophobic domains and consists predominantly of ,3-pleated sheet. These observations require the acommodation of polar and ionizable residues in an environment that has a low dielectric constant. Owing to a currently limited understanding of the constraints governing membrane protein structure, a miinimal approach to structure prediction is proposed that identifies segments causing polypeptides to reverse their direction (turn identification). The application of this procedure avoids hydrophobicity parameters and yields a model of porin which is in good agreement with all experimental data available. The presence of polar and ionizable residues within membrane boundaries implies a dense (saturating) network of hydrogen bond donor and acceptor groups. Application to a paradigm of hydrophobic membrane proteins, bacteriorhodopsin, reveals a pattern consistent with its a-helical folding. The postulated structure includes significantly more polar residues in the membrane domain than have been assumed previously, suggesting that there are also hydrogen bonding networks in bacteriorhodopsin. Extensive networks permeating protein interior and surfaces would explain the extraordinary stability and the tight interactions between functional units in the formation of crystaline arrays of both proteins.

The outer membrane phospholipase A (OMPLA) of Escherichia coli is present in a dormant state in the cell envelope. The enzyme is activated by various processes, which have in common that they perturb the outer membrane. Kinetic experiments, chemical cross-linking, and analytical ultracentrifugation were carried out with purified, detergent-solubilized OMPLA to understand the underlying mechanism that results in activation. Under conditions in which the enzyme displayed full activity, OMPLA was dimeric. High detergent concentrations or very dilute protein concentrations resulted in low specific activity of the enzyme, and under those conditions the enzyme was monomeric. The cofactor Ca 2؉ was required for dimerization. Covalent modification of the active site serine with hexadecylsulfonylfluoride resulted in stabilization of the dimeric form and a loss of the absolute calcium requirement for dimerization. The results of these experiments provide evidence for dimerization as the molecular mechanism by which the enzymatic activity of OMPLA is regulated. This dimerization probably plays a role in vivo as well. Data from chemical cross-linking on whole cells indicate that OMPLA is present in the outer membrane as a monomer and that activation of the enzyme induces dimerization concurrent with the appearance of enzymatic activity.Outer membrane phospholipase A (OMPLA 1 ; also known as detergent-resistant phospholipase A or PldA protein) is one of the few enzymes present in the outer membrane of Gramnegative bacteria. OMPLA hydrolyzes the acyl ester bonds in (phospho)lipids and has Ca 2ϩ as an essential cofactor (1, 2). Initially, the Escherichia coli enzyme was purified and characterized (3), and its structural gene, designated pldA, was subsequently cloned and overexpressed (4 -7). Recently, Brok et al. (8) reported the cloning of the pldA genes of several other Enterobacteriaceae species. Comparison of the OMPLA amino acid sequences revealed a high degree of homology within this family, but no clear homology exists with sequences of watersoluble (phospho)lipases. A ␤-barrel structure has been proposed for OMPLA (8), analogous to the outer membrane porins, of which the x-ray structures have been solved (9 -11). Recently, we succeeded in the overproduction, in vitro refolding, and subsequent purification of the enzyme on a large scale (12), which allowed its crystallization (13).Although OMPLA is embedded in its own substrate in the cell envelope, no enzymatic activity can be detected in normally growing cells (14,15). Because OMPLA is expressed constitutively, genetic regulation cannot explain the absence of enzymatic activity. Moreover, the expressed protein is correctly transported to and inserted into the outer membrane, where it resides in a dormant state. Activity is induced by various processes that perturb the membrane, such as phage-induced lysis (16), temperature shock (4), and colicin secretion (17, 18). Similar results have been reported in vitro after reconstitution of OMPLA in lipid vesicles (19). Memb...

In the tetragonal crystal form of OmpF porin, the membrane-exposed area is accessible from the aqueous solution. It is coated by a film of detergent molecules, which presumably mimics the interactions of the protein with lipids in the biological membrane. In the trigonal form, protein-protein interactions predominate in the hydrophobic zone. These may reflect the tight interactions between trimers that are observed in the biological membrane.