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Calculations of the minimum-energy conformations of cyclooctane, 1,3-dioxocane, and 1,3,6-trioxocane are presented. A potential energy function is determined that is consistent with the energies, geometries, and low-frequency vibrations of these rings. The vibrational study is presented in the following paper. A model is developed that provides a representation for the interconversion of the conformations of eight-membered rings. The boat-chair form is the minimum-energy conformer for all three molecules. In the case of the oxocanes, the oxygen atoms have definite preferred positions.

The association of Escherichia coli lac repressor with its operator and with nonoperator DNA has been extensively studied (for recent reviews, see refs. 1-3). Both the lac operator (4) and nonoperator DNA (5, 6) undergo structural changes as a consequence of repressor binding. The large bimolecular rate constant for association (7,8) implies that these changes occur within the repressor-DNA complex. We present evidence that the lac repressor undergoes at least two conformational changes as it binds to poly[d(A-T)]. Quite likely, one or more of these changes occurs simultaneously with changes in the DNA, serving to strengthen their interaction.The lac repressor was treated with N-(iodoacetylaminoethyl)-1-naphthylamine-5-sulfonate (I-AENS) (9) to covalently label cysteine residues with the fluorescent N-(acetylaminoethyl)-1-naphthylamine-5-sulfonate (AENS) group. Each repressor subunit has three cysteine residues (10) located in regions not thought to be directly involved in DNA binding (3). These residues display different reactivities toward the chromophoric reagents 2-chloromercuri-4-nitrophenol and 2-bromoacetamido-4-nitrophenol (11). However, the fluorescent reagent fluorescein mercuric acetate shows little selectivity among the three sulfhydryls (12). We find that I-AENS reacts selectively with one cysteine residue in each repressor subunit. Stopped-flow techniques have been used to monitor the events during the association of repressor with poly[d(A-T)] that alter the fluorescence of this AENS label.MATERIALS AND METHODS Materials. The lac repressor was isolated from E. coli CSH 46 and assayed for operator binding activity as described (13). Other materials were obtained from the following sources:poly[d(A-T)] from Miles, I-AENS from Aldrich, iodo[14C]-acetamide from New England Nuclear, and trypsin (TRL) and chymotrypsin (CDI) from Worthington.Labeling of lac Repressor. Repressor (1-3 mg/ml) in 0.2 M Tris-HCI (pH 8.6 at 40C), 0.5 M KCI, and 0.1 mM EDTA, was reacted with I-AENS (usually 8.1 mM) at 40C in the dark. At the appropriate time, the protein was separated from excess reagent by gel filtration through Sephadex G-25 and then dialyzed overnight against the buffer used for labeling, with 0.1 mM dithiothreitol added. The concentration of repressor and extent of AENS labeling were determined from the absorbance at 280 and 340 nm by using the following values: lac repressor subunit, E2W = 22,125 M-1 cm-1 (14), Ec40 = 105 M-1 cm-1; AENS, E280 = 1260 M-1 cm-1, E3O = 6850 M-1 cm-1 (9).Identification of Labeled Cysteine Residues. AENS-labeled repressor was reacted with iodo[14C]acetamide (1.68 Ci/mole, 1 Ci = 3.70 X 1010 becquerels) in 8 M urea at 320C. The molar concentration of iodoacetamide was 6 times that of repressor subunits. After 30 min, excess iodoacetamide was reacted with dithiothreitol, and the splution was dialyzed against 0.1 M NH4HCO3/2 M urea. This protein was then digested with trypsin (1% wt/wt) for 2.5 hr at 250C. A second addition of trypsin was followed by an additional 2.5-hr digestion....

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