UNIFORM N-15 LABELING OF A FUNGAL PEPTIDE - THE STRUCTURE AND DYNAMICS OF AN ALAMETHICIN BY N-15 AND H-1-NMR SPECTROSCOPY

An alamethicin, secreted by the fungus Trichoderma viride and containing a glutamine at position 18 instead of the usual glutamic acid, has been uniformly labeled with N-15 and purified by HPLC. The extent of N-15 incorporation at individual backbone and side-chain sites was found to vary from 85% to 92%, as measured by spin-echo difference spectroscopy. The proton NMR spectrum of the peptide dissolved in methanol was assigned using correlation spectroscopies and nuclear Overhauser enhancements (NOE) measured in the rotating frame. The N-15 resonances were assigned by the 2D H-1-N-15 correlation via heteronuclear multiple-quantum coherence experiment. NOEs and 3J(NHC-alpha-H) coupling constants strongly suggest that, in methanol, from Aib-3 to Gly-11, the peptide adopts a predominantly helical conformation, in agreement with previous H-1 NMR studies [Esposito, G., Carver, J. A., Boyd, J., & Campbell, I.D. (1987) Biochemistry 26,1043-1050; Banerjee, U., Tsui, F.-P., Balasubramanian, T. N., Marshall, G. R., & Chan, SI. (1983) J. Mol. Biol. 165, 757-7751. The conformation of the carboxyl terminus (12-20) is less well determined, partly because the amino acid composition reduces the number of NOEs and coupling constants which can be determined by H-1 NMR spectroscopy. The 3J(NHC-alpha-H) in the C-terminus suggest the possibility of conformational averaging at Leu-12, Val-15, and Gln-19, an interpretation which is supported by a recent molecular dynamics simulation of the peptide [Fraternalli, F. (1990) Biopolymers 30, 1083-10991. The dynamics at each side-chain and backbone nitrogen were measured by the heteronuclear N-15{H-1} NOE, but these measurements could not confirm a model in which the carboxyl terminus experiences greater conformational freedom than the amino terminus.