N-15 NMR RELAXATION STUDIES OF CALCIUM-LOADED PARVALBUMIN SHOW TIGHT DYNAMICS COMPARED TO THOSE OF OTHER EF-HAND PROTEINS

Dynamics of the rat cl-parvalbumin calcium-loaded form have been deter mined by measurement of N-15 nuclear relaxation using proton-detected heteronuclear NMR spectroscopy. The relaxation data were analyzed usin g spectral density functions and the Lipari-Szabo formalism. The major dynamic features for the rat alpha-parvalbumin calcium-loaded form ar e (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 pro tein being restricted in their mobility, (3) a conformational exchange occurring at the kink of helix D, and (4) the residue at relative pos ition 2 in the Ca2+-binding sites having an enhanced mobility. Compari son of the Ca2+-binding EF-hand domains of alpha-parvalbumin-Ca2+, cal bindin-Ca2+, and calmodulin-Ca2+ shows that parvalbumin is probably th e most rigid of the EF-hand proteins. It also illustrates the dynamica l 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 res tricted mobility for the other residues in the binding loop, and (3) a n overall rigidity for the helices of EF-hand motifs. The differences in mobility between parvalbumin and the two EF-hand proteins occur mai nly at the linker connecting the pair of EF hands and also at the C te rminus of the last helix. In parvalbumin-Ca2+, these two regions are c haracterized by a pronounced rigidity compared to the corresponding mo re mobile regions in calbindin-Ca2+ and calmodulin-Ca2+.