Solvation energetics and conformational change in EF-hand proteins.
Calmodulin and other members of the EF-hand protein family are known to undergo major changes in conformation upon binding Ca(2+). However, some EF-hand proteins, such as calbindin D9k, bind Ca(2+) without a significant change in conformation. Here, we show the importance of a precise balance of solvation energetics to conformational change, using mutational analysis of partially buried polar groups in the N-terminal domain of calmodulin (N-cam). Several variants were characterized using fluorescence, circular dichroism, and NMR spectroscopy. Strikingly, the replacement of polar side chains glutamine and lysine at positions 41 and 75 with nonpolar side chains leads to dramatic enhancement of the stability of the Ca(2+)-free state, a corresponding decrease in Ca(2+)-binding affinity, and an apparent loss of ability to change conformation to the open form. The results suggest a paradigm for conformational change in which energetic strain is accumulated in one state in order to modulate the energetics of change to the alternative state.
Ababou
A
A
Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Desjarlais
J R
JR
D000595:Amino Acid Sequence; D064026:Calbindins; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry*; D002942:Circular Dichroism; D004252:DNA Mutational Analysis; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D064030:S100 Calcium Binding Protein G / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D064026:Calbindins; D002147:Calmodulin; D011994:Recombinant Proteins; D064030:S100 Calcium Binding Protein G; C576506:S100G protein, human; D002118:Calcium