Proton NMR studies of a metallothionein from Neurospora crassa: sequence-specific assignments by NOE measurements in the rotating frame

Abstract

Sequential 1H NMR assignments of metallothionein from Neurospora crassa have been accomplished by the combined use of COSY, 2QF-COSY, HOHAHA, and rotating-frame NOE experiments. All potentially observable resonances were assigned except for the .epsilon.-NH3 group of the C-terminal lysine. 1H NOEs, when observed in the laboratory frame and at 500-MHz spectrometer frequency, were negligible in this protein due to the inherent rotational correlation time of the molecule. This difficulty was circumvented by measuring transverse NOEs in the rotating frame under spin-locking conditions. The observed pattern of NOEs reveals a marked absence of "regular'' secondary structures in the protein. Thus, the stability of this metallothionein''s tertiary structure must arise primarily for its metal ligation. This appears to be a general feature of MTs since a general lack of extensive secondary structural elements was also observed in other metallothioneins.