Assignment of 13C Resonances and Analysis of Relaxation Properties and Internal Dynamics of Pike Parvalbumin by 13C‐NMR at Natural Abundance

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Abstract
Pike parvalbumin is an 11.5-kDa globular protein which binds Ca2+ through EF-hand structural motifs. Nearly complete assignment of the protonated 13C resonances has been achieved by means of heteronuclear two-dimensional experiments. The study shows that 13Ca chemical shifts can be very sensitive to localised conformational aspects. To characterise internal dynamics of pike parvalbumin, longitudinal-relaxation and transverse-relaxation rates and 1H-13C NOEs were measured for alpha-carbons at natural abundance by means of two-dimensional NMR spectroscopy. Relaxation data were obtained at a spectrometer frequency of 600 MHz for 69 residues with an even spread along the parvalbumin polypeptide chain. A double approach that included Lipari-Szabo analysis and direct mapping of spectral densities was used to interpret relaxation data in terms of internal dynamics. The former analysis provides valuable information about the overall rotational correlation time and S2 order parameters, while the mapping approach characterises the relative contributions of different motional frequencies. The results suggest that Ca(2+)-loaded pike parvalbumin has a rigid structure, even in the functional regions, i.e., the Ca(2+)-binding loops. The patterns of density-function values are more sensitive to the secondary structure than those of S2. Moreover, depending on the sampling frequency, these patterns reveal different aspects of structure-specific motions.