15N NMR Relaxation Studies of Calcium-Loaded Parvalbumin Show Tight Dynamics Compared to Those of Other EF-Hand Proteins

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Abstract
Dynamics of the rat α-parvalbumin calcium-loaded form have been determined by measurement of 15N nuclear relaxation using proton-detected heteronuclear NMR spectroscopy. The relaxation data were analyzed using spectral density functions and the Lipari−Szabo formalism. The major dynamic features for the rat α-parvalbumin calcium-loaded form are (1) the extreme rigidity of the helix−loop−helix EF-hand motifs and the linker segment connecting them, (2) the N and C termini of the protein being restricted in their mobility, (3) a conformational exchange occurring at the kink of helix D, and (4) the residue at relative position 2 in the Ca2+-binding sites having an enhanced mobility. Comparison of the Ca2+-binding EF-hand domains of α-parvalbumin−Ca2+, calbindin−Ca2+, and calmodulin−Ca2+ shows that parvalbumin is probably the most rigid of the EF-hand proteins. It also illustrates the dynamical properties which are conserved in the EF-hand domains from different members of this superfamily: (1) a tendency toward higher mobility of NH vectors at relative position 2 in the Ca2+-binding loop, (2) a restricted mobility for the other residues in the binding loop, and (3) an overall rigidity for the helices of EF-hand motifs. The differences in mobility between parvalbumin and the two EF-hand proteins occur mainly at the linker connecting the pair of EF hands and also at the C terminus of the last helix. In parvalbumin−Ca2+, these two regions are characterized by a pronounced rigidity compared to the corresponding more mobile regions in calbindin−Ca2+ and calmodulin−Ca2+.