Growth of two-dimensional streptavidin crystals at the air-water interface has been used to study protein molecular interactions at various pH values. Between pH 5 and 6, several unique crystal domain shapes are visible with fluorescence microscopy. Characterization of a chiral domain with transmission electron microscopy reveals a composition of two coexisting crystal types. The bulk crystal, one previously seen at pH 4 (space group P1) (Hemming et al. J. Mol. Biol. 1995, 246, 308), has interspersed within it at approximately 5% aerial extent a new crystal type with unit cell parameters a ) 116 Å, b ) 58 Å, and γ ) 107°(space group P2). This new form exists in the chiral domains as long narrow crystals and is characterized by relatively weak and anisotropic molecular interactions. The packing arrangement for the new P2 crystal at pH 5-6 exhibits characteristics of the pH 4 crystal and of the C222 crystal (Darst et al. Biophys. J. 1991, 59, 387) obtained at pH 7.

Total internal reflection fluorescence microscopy (TIRFM) has been combined with functional reconstitution of the mouse IgG receptor moFc gamma RII in substrate-supported planar membranes to quantitatively probe IgG-moFc gamma RII interactions. MoFc gamma RII was purified from the macrophage-related cell line J774A.1 using affinity chromatography with Fab fragments of the anti-moFc gamma RII monoclonal antibody 2.4G2. Purified moFc gamma RII was reconstituted into liposomes by detergent dialysis, and the liposomes were fused on quartz substrates to form supported planar membranes containing moFc gamma RII. TIRFM measurements showed that fluorescently labeled 2.4G2 Fab specifically bound to the planar membranes, confirming the presence of moFc gamma RII. The receptor density in the planar membranes was sufficiently high to allow direct detection of bound, fluorescently labeled polyclonal and monoclonal mouse IgG with TIRFM, demonstrating that moFc gamma RII retained Fc-mediated IgG binding activity after planar membrane formation and permitting direct measurement of bound IgG as a function of the IgG solution concentration. Cross-inhibition measurements showed that polyclonal mouse IgG blocked the binding of labeled 2.4G2 Fab and that 2.4G2 Fab blocked the binding of labeled polyclonal IgG. This work provides a direct measure of the relatively weak IgG-moFc gamma RII association constant and demonstrates a new model system in which the chemical and physical properties of IgG-moFc gamma RII interactions can be quantitatively characterized as a function of membrane, antibody, and solution properties.

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A procedure for constructing substrate-supported planar membranes using membrane fragments isolated from the macrophage-related cell line J774A.1 is described. Total internal reflection (TIR) fluorescence microscopy is employed to demonstrate that fluorescently labeled Fab fragments of a monoclonal antibody (2.4G2) with specificity for a murine macrophage cell-surface receptor for IgG (moFc gamma RII) bind to the planar model membranes. These measurements show that the planar membranes contain moFc gamma RII and yield a value for the association constant of 2.4G2 Fab fragments with moFc gamma RII equal to (9.6 +/- 0.4) x 10(8) M-1 and indicate that the surface density of reconstituted moFc gamma RII is approximately 50 molecules/microns 2. In addition, TIR fluorescence microscopy is used to investigate the Fc-mediated competition of unlabeled, polyclonal murine IgG with labeled 2.4G2 Fab fragments for moFc gamma RII in the planar membranes. These measurements indicate that the reconstituted moFc gamma RII recognized by 2.4G2 Fab fragments also retains the ability to bind murine IgG Fc regions and yield a value for the association constant of polyclonal murine IgG with moFc gamma RII equal to (1-5) x 10(5) M-1. This work represents one of the first applications of TIR fluorescence microscopy to specific ligand-receptor interactions.

The structure of an actively transcribing complex, containing yeast RNA polymerase II with associated template DNA and product RNA, was determined by electron crystallography. Nucleic acid, in all likelihood the "transcription bubble" at the active center of the enzyme, occupies a previously noted 25 A channel in the protein structure. Details are indicative of a roughly 90 degrees bend of the DNA between upstream and downstream regions. The DNA apparently lies entirely on one face of the polymerase, rather than passing through a hole to the opposite side, as previously suggested.