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¹⁵N nuclear magnetic resonance relaxation studies on rat ß-parvalbumin and the pentacarboxylate variants, S55D and G98D

¹ Department of Biochemistry, University of Missouri–Columbia, Columbia, Missouri 65211, USA
² Department of Chemistry, University of Missouri–Columbia, Columbia, Missouri 65211, USA
³ Discovery, Pharmacia Corp., Chesterfield, Missouri 63198, USA

Reprint requests to: Dr. Michael T. Henzl, Department of Biochemistry, University of Missouri–Columbia, Columbia, Missouri 65211; e-mail: henzlm{at}missouri.edu; fax: (573) 884-4812.

¹⁵N relaxation data for Ca²⁺-bound rat ß-parvalbumin (a.k.a. oncomodulin) were analyzed using the Lipari-Szabo formalism and compared with existing data for rat -parvalbumin. Although the average S² values for the two proteins are very similar (0.85 for , 0.84 for ß), residue-by-residue inspection reveals systematic differences. tends to have the lower S² value in helical regions; ß tends to have the lower value in the loop regions. Rat ß was also examined in the Ca²⁺-free state. The 59 assigned residues displayed an average order parameter (0.90) significantly greater than the corresponding residues in the Ca²⁺-loaded form. The pentacarboxylate variants of rat ß—S55D and G98D—also were examined in the Ca²⁺-bound state. Although both mutations significantly heighten Ca²⁺ affinity, they utilize distinct energetic strategies. S55D improves the Ca²⁺-binding enthalpy; G98D improves the binding entropy. They also show disparate peptide backbone dynamics. Whereas ß G98D displays an average order parameter (0.87) slightly greater than that of the wild-type protein, ß S55D displays an average order parameter (0.82) slightly lower than wild-type ß. Furthermore, whereas just two backbone N-H bonds in ß G98D show internal motion on the 20–200-psec timescale, fully 52 of the 93 residues analyzed in ß S55D show this behavior. These findings suggest that the increased electrostatic repulsion attendant to introduction of an additional carboxylate into the CD site ligand array impedes backbone vibrational motion throughout the molecule.

Keywords: Calcium-binding proteins; EF-hand proteins; parvalbumins; NMR; dynamics

Abbreviations: CD site, parvalbumin Ca²⁺-binding site spanning residues 41–70 and including the C and D helices • EF site, parvalbumin Ca²⁺-binding site spanning residues 80–108 and including the E and F helices • Hepes, 4–(2-hydroxyethyl)-1-piperazinesulfonic acid • HSQC, heteronuclear single quantum coherence • NMR, nuclear magnetic resonance • NOESY, nuclear Overhauser effect spectroscopy • PV, parvalbumin • TOCSY, total correlated spectroscopy.

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