ASSIGNMENT OF C-13 RESONANCES AND ANALYSIS OF RELAXATION PROPERTIES AND INTERNAL DYNAMICS OF PIKE PARVALBUMIN BY C-13-NMR AT NATURAL-ABUNDANCE

Pike parvalbumin is an 11.5-kDa globular protein which binds Ca2+ thro ugh EF-hand structural motifs. Nearly complete assignment of the proto nated C-13 resonances has been achieved by means of heteronuclear two- dimensional experiments. The study shows that C-13(alpha) chemical shi fts can be very sensitive to localised conformational aspects. To char acterise internal dynamics of pike parvalbumin, longitudinal relaxatio n and transverse-relaxation rates and H-1-C-13 NOEs were measured for alpha-carbons at natural abundance by means of two-dimensional NMR spe ctroscopy. Relaxation data were obtained at a spectrometer frequency o f 600 MHz for 69 residues with an even spread along the parvalbumin po lypeptide chain. A double approach that included Lipari-Szabo analysis and direct mapping of spectral densities was used to interpret relaxa tion data in terms of internal dynamics. The former analysis provides valuable information about the overall rotational correlation time and S-2 order parameters, while the mapping approach characterises the re lative contributions of different motional frequencies. The results su ggest that Ca2+-loaded pike parvalbumin has a rigid structure, even in the functional regions, i.e., the Ca2+-binding loops. The patterns of density-function values are more sensitive to the secondary structure than those of S-2. Moreover, depending on the sampling frequency, the se patterns reveal different aspects of structure-specific motions.