CONFORMATIONAL DIVERSITY AND DIFFERENTIAL TYROSYL MOTIONS IN MET(5)-ENKEPHALINS AND LEU(5)-ENKEPHALINS AND RELATED PEPTIDES AS STUDIED BY HIGH-RESOLUTION SOLID-STATE C-13 NMR-SPECTROSCOPY

We have recorded C-13 CP-MAS NMR spectra of Leu(5)- and Met(5)-enkepha lins crystallized from a variety of solvent systems. It was found that the secondary structure of the enkephalins is well related to the man ner of the peak-splitting in Tyr C, signals; the two distinct peaks we re observed for the extended forms of Met(5)- and Leu(5)-enkephalins, whereas a broad single signal was observed for Leu(5)-enkephalin takin g the P-bend form. We found that C-13 NMR spectra of freshly prepared Leu(5)-enkephalin mono- or trihydrate differ from those of samples con tained in a rotor without airtight sealing. This was caused by irrever sible conformational change owing to dehydration during magic angle sp inning. In this connection, it was shown that conformational diversity of crystalline enkephalins was well characterized by C-13 NMR spectra . It is demonstrated that C-13 NMR spectra of the trihydrate recorded below -40 degrees C lead to an additional spectral change indicating t he doubling of the peaks in Tyr C-xi and Phe C-delta peaks caused by t he presence of conformational isomerism about the C-alpha-C-beta (defi ned by (chi 1) angle). The spectral profile of Tyr C-epsilon was well related to the presence or absence (or more precisely to the rate cons tant) of the flip-flop motion of the tyrosine side chain because the b road single peak is changed into a well-defined doublet peak when the sample was cooled down to -80 degrees C. The rate constant for such fl ip-flop motion was estimated as 1.3 x 10(2) s(-1) at ambient temperatu re based on a spectral simulation utilizing the two-site exchange mode l. We further recorded one- or two-dimensional exchange spectra in ord er to analyze the similar flip-flop motions whose rate constants are m uch smaller than the limiting value as estimated from the simple line- shape analysis of the two-site exchange (10 s(-1)). The rate constant for the flip-flop motion of Ac-Tyr-NH2 and the extended form of Met(5) -enkephalin are found to be 1.94 and 1.45 s(-1) at ambient temperature , respectively. We were also able to determine the rate constants of v ery slow flip-flop motions from an order of magnitude of 1 through 10( -3) s(-1) in the cases of Tyr-OH or Tyr-NH2. The present observations indicate that a detailed analysis of the flip-flop motions of the Tyr side chain provides an invaluable clue for analyzing the manner of mol ecular packing or flexibility of peptides.