POSITIVE PHI-ANGLES IN PROTEINS BY NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY

Non-glycine residues with positive phi-angles have been identified in four proteins, barley serine proteinase inhibitor CI-2, bacterial ribonuclease (barnase) of Bacillus amyloliquefaciens, hen egg white lysozyme and a basic protein from barley seed (barwin) by use of nuclear magnetic resonance spectroscopy. By accurate measurements of the coupling constant 3J(H(N)H(alpha)). and integration of the nuclear Overhauser H(N)-H(alpha) cross peak, positive phi-angles could be determined reliably to 60-degrees +/- 30-degrees, in full agreement with the crystal structures for lysozyme, barnase and serine proteinase-inhibitor CI-2. The work emphasizes that positive phi-angles can also occur in non-glycine residues and in the four proteins, positive phi-angles have been observed for the residue types aspartic acid, asparagine, arginine, serine, glutamine, histidine, tyrosine, tryptophan and phenylalanine. The measured 3J(H(N)H(alpha)) coupling constants and the intensity of the intraresidue H(N)-H(alpha) NOEs agree well with the solution structures of three of the proteins, using the existing parametrization of the Karplus curve (Pardi, A., Billeter, M. and Wuthrich, K. (1984) J. Mol. BioL, 180, 741-75 1; Ludvigsen, S., Andersen, K.V. and Poulsen, F. M. (1991) J. Mol. Biol., 217, 731-736).