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The structure of an early M-intermediate of the wild-type bacteriorhodopsin photocycle formed by actinic illumination at 230 K has been determined by x-ray crystallography to a resolution of 2.0 A. Three-dimensional crystals were trapped by illuminating with actinic light at 230 K, followed by quenching in liquid nitrogen. Amide I, amide II, and other infrared absorption bands, recorded from single bacteriorhodopsin crystals, confirm that the M-substate formed represents a structure that occurs early after deprotonation of the Schiff base. Rotation about the retinal C13-C14 double bond appears to be complete, but a relatively large torsion angle of 26 degrees is still seen for the C14-C15 bond. The intramolecular stress associated with the isomerization of retinal and the subsequent deprotonation of the Schiff base generates numerous small but experimentally measurable structural changes within the protein. Many of the residues that are displaced during the formation of the late M (M(N)) substate formed by three-dimensional crystals of the D96N mutant (Luecke et al., 1999b) are positioned, in early M, between their resting-state locations and the ones which they will adopt at the end of the M phase. The relatively small magnitude of atomic displacements observed in this intermediate, and the well-defined positions adopted by nearly all of the atoms in the structure, may make the formation of this structure favorable to model (simulate) by molecular dynamics.

When purified without the use of ionic detergents, both OmpA and OprF proteins contained nearly 20% ␣-helical structures, which disappeared completely upon the addition of sodium dodecyl sulfate. This result suggests that the proteins fold in a similar manner, with an N-terminal, membrane-spanning ␤-barrel domain and a C-terminal, globular, periplasmic domain.OmpA is one of the major outer membrane proteins of Escherichia coli. It appears to exist as a monomer, and it shows very little pore-forming activity (16), apparently because only a small fraction of the OmpA population contains open channels (17). The accepted folding model of OmpA contains an Nterminal domain (residues 1 to 170) consisting of eight antiparallel ␤-strands, as well as a periplasmic C-terminal domain (residues 196 to 325) (8, 12). The two domains are connected by an Ala-Pro-rich hinge sequence. The proposed structure of the N-terminal domain is supported by a wealth of data, including data indicating the localization of phage-binding sites exclusively on segments predicted to correspond to the external loops connecting ␤-strands (8) and Raman spectroscopy data (19). However, the secondary structure of the C-terminal domain has so far received little attention.Pseudomonas aeruginosa outer membrane contains OprF as a major protein. It is a homolog of E. coli OmpA (2) and similarly produces diffusion pores of low permeability (5,9,20). In contrast to OmpA, however, the entire length of OprF has been proposed to traverse the outer membrane as 18 ␤-strands (11).We studied the secondary structure of these proteins, which were purified in the total absence of denaturing, ionic detergents, in view of these conflicting models of folding. OmpA was purified as follows. Cell envelope fraction from a 4-liter culture of HN705 (⌬ompC ompF::Tn5) (16) was first extracted twice with 50-ml portions of 20 mM N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic acid (HEPES)-Na buffer (pH 7.5)-3% octylpolyoxyethylene (POE) (Alexis Biochemicals, San Diego, Calif.) (10) and then extracted once with 50 ml of the same solution containing 1 M NaCl in addition to solubilize most of the cytoplasmic membrane proteins. The residue was then extracted with 20 ml of 20 mM HEPES buffer-10% octyl-POE-1 M NaCl, and the extract was fractionated by gel filtration on a column (1.5 by 92 cm) of Toyo Pearl 50F (TosoHaas, Montgomeryville, Pa.), equilibrated, and eluted with 0.1% dodecyl maltoside-0.4 M NaCl-10 mM HEPES buffer-3 mM NaN 3 . When the circular dichroism (CD) spectrum was taken on a sample concentrated by ultrafiltration through an Amicon (Beverly, Mass.) PM-10 filter, it showed a strong ellipticity with two minima at 209 and 222 nm, characteristic of structures containing a significant fraction of ␣-helix. When curve fitting was used to estimate the secondary structure (1), OmpA appeared to contain 18% ␣-helix, 40% ␤-strand, 5% ␤-turn, and 25% unordered structure (Fig. 1). In contrast, OmpA prepared by solubilization of the outer membrane by sodium dodecyl sulfate (SDS) (16) ...