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Polytheonamide B (pTB), a highly cytotoxic polypeptide, is one of the most unusual nonribosomal peptides of sponge origin. pTB is a linear 48-residue peptide with alternating D- and L-amino acids and contains a total of eight types of nonproteinogenic amino acids. To investigate the mechanisms underlying its cytotoxic activity, we determined the three-dimensional structure of pTB by NMR spectroscopy, structure calculation, and energy minimization. pTB adopts a single right-handed β(6.3)-helical structure in a 1:1 mixture of methanol/chloroform with a length of approximately 45 A and a hydrophilic pore of ca. 4 A inner diameter. These features indicate that pTB molecules form transmembrane channels that permeate monovalent cations as gramicidin A channels do. The strong cytotoxicity of pTB can be ascribed to its ability to form single molecule channels through biological membranes.

Purple membrane suspension shows two different orientations in electric fields of different frequencies. The orientation at low frequencies (less than or equal to approximately 10 Hz), with the membrane surface perpendicular to the electric field, is due to permanent dipole moment of the membrane and the orientation at high frequencies (greater than or equal to approximately 100 Hz), with the surface parallel to the electric field, is due to induced dipole moment. By quantitative analysis of these orientations, we determined the permanent dipole moment and the polarizability. Both values varied according to the membrane size: the permanent dipole moment ranged from 500 kD to 10 MD and was proportional to the square of the diameter of the membrane. The polarizability ranged from 1 X 10(-13) to 1 X 10(-11)cm3 and was proportional to the third to fourth power of the diameter. Because the permanent dipole moment was proportional to the area of the membrane, we could determine permanent dipole moment per bacteriorhodopsin. By determining the actual membrane size under electron microscopy, we got 98 D/bacteriorhodopsin. We also concluded that the direction of the permanent dipole moment was from the cytoplasmic to the extracellular side. These values, however, were strongly dependent on the ionic strength in the medium, suggesting a screening effect due to counter ions near the membrane surface. We evaluated the screening effect and showed about a four-charge difference between the two sides of the purple membrane. Under illumination, we found that the permanent dipole moment decreased from 98 to 63 D/bacteriorhodopsin. From the best-oriented sample, we also concluded that the angle of retinal against the axis normal to the membrane surface was greater than 68.6 degrees.

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