CALCIUM-BINDING MECHANISM OF HUMAN NONERYTHROID ALPHA-SPECTRIN EF-STRUCTURES

We have used circular dichroism and H-1- and N-15-NMR spectroscopy to investigate calcium binding to the two EF-hands of human nonerythroid or alpha II-spectrin. Comparison of the H-1-NMR spectra from the pepti de containing both EF-hands to the peptides containing the single EF-I and EF-II structures showed that both the structural and calcium-bind ing 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 s hifts changes were recorded using multidimensional NMR spectroscopy du ring calcium titration. A total of 25 titration curves were obtained, each corresponding to the chemical shift changes of individual amino a cid residues. The shapes of these titration curves were either hyperbo lic or sigmoidal. Using factor analysis, two functions were extracted, one hyperbolic and one sigmoidal, which accounted for nearly all info rmation 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 sigmoida l type was more pronounced in the titration curves corresponding to re sidues from the first EF-hand, we suggest that calcium binding to the first EF-hand is described by the sigmoidal function, and that the hyp erbolic function describes calcium binding to the second EF-hand. Ther efore, is seems likely that the second EF-hand must contain bound calc ium before the first EF-hand can bind.