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The hydrophobic effect is the main thermodynamic driving force in the folding of water-soluble proteins. Exclusion of nonpolar moieties from aqueous solvent results in the formation of a hydrophobic core in a protein, which has been generally considered essential for specifying and stabilizing the folded structures of proteins. Outer surface protein A (OspA) from Borrelia burgdorferi contains a three-stranded beta-sheet segment which connects two globular domains. Although this single-layer beta-sheet segment is exposed to solvent on both faces and thus does not contain a hydrophobic core, the segment has a high conformational stability. Here we report the engineering of OspA variants that contain larger single-layer beta-sheets (comprising five and seven beta-strands) by duplicating a beta-hairpin unit within the beta-sheet. Nuclear magnetic resonance and small-angle X-ray scattering analyses reveal that these extended single-layer beta-sheets are formed as designed, and amide hydrogen-deuterium exchange and chemical denaturation show that they are stable. Thus, interactions within the beta-hairpin unit and those between adjacent units, which do not involve the formation of a hydrophobic core, are sufficient to specify and stabilize the single-layer beta-sheet structure. Our results provide an expanded view of protein folding, misfolding and design.

Vol. 66 lated: 0.31 g. of VIII (63%); 0.19 g. of II (35%); 0.20 g. of salicylic acid and traces of carbomethoxysalicylic acid. One gram of the ester under the same conditions yielded 0.18 g. of VIII after refluxing for five minutes.3,3' -Methylenebis -(4 -hydroxycoumarin) dibenzoate (1.12 g.) in 12 ml. of absolute ethanol was refluxed with 2 moles of sodium ethoxide for fifteen hours. Two moles of 1 N hydrochloric acid was added and the combined precipitate of II and VIII was filtered off and II separated from VIII by means of its solubility in dilute NaOH. VIII (0.13 g.) m. p. 318-320°and II (0.56 g.) m. p. 290°w ere obtained. One mole of NaHCOs was added to the filtrate and after concentration to dryness in vacuo, the residue was acidified and extracted with ether. The acid fraction yielded 0.12 g. of benzoic acid, m. p. 118-120°, and the neutral solution yielded 0.02 g. of ethyl benzoate, b. p. 206-207°.3,3'-Methylenebis-(4-hydroxycoumarin) Disalicylate.-The di-(o-benzyloxybenzoate) of I (3.9 g.) in 200 ml. of dioxane was hydrogenated at 1900 pounds pressure and 100°for three hours over Raney nickel. The catalyst was filtered off and the filtrate concentrated to dryness in vacuo.The residue was taken up in a small volume of hot dioxane and the insoluble residue of II was filtered from the hot solution. This process was repeated three times. After a final recrystallization from acetic acid the disalicylate.of II melted at 223-225°, yield 0.7 g.

1. The hemorrahgic agent is spoiled sweet clover hay (Melilotus alaba) has been isolatd in a pure state, m. p. 288‐289°, from experimentally produced spoiled hays as well as form hyas that killed cattle in argicultural practice. 2. This substance has the empirical formula C12H12O6. It is optically inactive. There are two acidic hydroxyls in the molecule. The acidity of the pure substance falls between that of the phenols and the carboxylic acids. A crystalline dimethyl other C12H12O4(OCH2)2 with a melting point fo 168‐170° (physiologically inactive) has been prepared by methylation with diazomethane. 3. In the pure state the substance has a low soulubility in the ordinary organic solvents. It is insoluble in acid media. Basic solvenets. and dilute alkali effect solution radily (salt formation). 4. The substance could not be characterized or identified on the basis of its behavior towards the usual identification reagents. Its occurrence in nature has not previosly been reproted. 5. 1.5 mg. of hte crystalline hemorrhagic agent cause approximately the same reduciton in the prothrombin level or activity of the plasma of standardized susceptible reabbits in 40 hours as 50 gm. of the standard spoiled hay sample. 6. Spoiled sweet cover hays produced experimentally from Melilotus alba and those relized in agricultural parctice contain approximately 0.003 per cent of the hemorrhagic agent on the dry substance basis. The over‐all yield of the subsance in a fractionation scheme involving sixteen steps approximates 86 per cent of the quantity present.