Abstract
High resolution natural abundance carbon‐13 NMR has been used previously to examine molecular motions and conformational transitions in the muscle calcium binding parvalbumins isolated from mirror carp. [S. J. Opella, D. J. Nelson, and O. Jardetzky, J. Chem. Phys. 64, 2533 (1976) and D. J. Nelson, S. J. Opella, and O. Jardetzky, Biochemistry 15, 5552 (1976).] The carbon‐13 NMR spectrum of parvalbumin typically reveals the presence of a number of well‐resolved resonances from single‐carbon sites in the protein. Since accurate assignment of these resonances to specific carbon atoms in the protein is essential before these resonances can be employed as probes of local conformational events, an investigation of the origin of a number of single‐carbon resonances has been performed. Carbon‐13 NMR on Tb(III)‐ and Yb(III)‐substituted parvalbumin is the principal spectroscopic technique employed; however, results from terbium fluorescence spectroscopy, γ‐ray scintillation spectroscopy, and x‐ray difference Fourier analysis all contribute to single‐carbon site assignment. The principal conclusions drawn from the combined spectroscopic results are that: (1) the downfield carboxyl resonance at 184.6 ppm, previously attributed to Glu‐81, whose carboxyl group is involved in an internal ionic bond, originates rather from a carboxyl function coordinating the solvent exposed metal ion, (2) the carbonyl resonance at 168.9 ppm arises from Lys‐96, as was previously suggested; and (3) the 11.2 ppm resonance derives from the δ‐methyl carbon of Ile‐97, a resonance assignment that could not be made previously.