Calcium-binding mechanism of human nonerythroid alpha-spectrin EF-structures.
We have used circular dichroism and 1H- and 15N-NMR spectroscopy to investigate calcium binding to the two EF-hands of human nonerythroid or alphaII-spectrin. Comparison of the 1H-NMR spectra from the peptide containing both EF-hands to the peptides containing the single EF-I and EF-II structures showed that both the structural and calcium-binding properties are significantly different. Further studies of the 121 amino acid peptide containing both EF-hands using circular dichroism and NMR showed that the binding of calcium ions induces conformational changes. To investigate the calcium-binding mechanism, the chemical shifts changes were recorded using multidimensional NMR spectroscopy during calcium titration. A total of 25 titration curves were obtained, each corresponding to the chemical shift changes of individual amino acid residues. The shapes of these titration curves were either hyperbolic or sigmoidal. Using factor analysis, two functions were extracted, one hyperbolic and one sigmoidal, which accounted for nearly all information present in the titration curves. By fitting the two functions to binding curves based on different binding models, we found that the binding mechanism is best described as sequential. Since the sigmoidal type was more pronounced in the titration curves corresponding to residues from the first EF-hand, we suggest that calcium binding to the first EF-hand is described by the sigmoidal function, and that the hyperbolic function describes calcium binding to the second EF-hand. Therefore, is seems likely that the second EF-hand must contain bound calcium before the first EF-hand can bind.
Lundberg
S
S
Department of Biochemistry, Umeå University, Sweden.
Buevich
A V
AV
Sethson
I
I
Edlund
U
U
Backman
L
L
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D012815:Signal Processing, Computer-Assisted; D013049:Spectrin / Q000737:chemistry / Q000378:metabolism*
D010446:Peptide Fragments; D011994:Recombinant Proteins; D013049:Spectrin; D002118:Calcium