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Biochemistry. 2005 Jan 25;44(3):905-14.

Dynamic motion of helix A in the amino-terminal domain of calmodulin is stabilized upon calcium activation.

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Cell Biology and Biochemistry Group, Biological Sciences Division, Fundamental Sciences Directorate, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, USA.


Calcium-dependent changes in the internal dynamics and average structures of the opposing globular domains of calmodulin (CaM), as well as their relative spatial arrangement, contribute to the productive association between CaM and a range of different target proteins, affecting their functional activation. To identify dynamic structural changes involving individual alpha-helical elements and their modulation by calcium activation, we have used site-directed mutagenesis to engineer a tetracysteine binding motif within helix A near the amino terminus of calmodulin (CaM), permitting the selective and rigid attachment of the fluorescent probe 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH) with full retention of function. The rigid tetracoordinate linkage of FlAsH to CaM, in conjunction with frequency domain fluorescence anisotropy measurements, allows assessment of dynamic changes associated with calcium activation without interference from independent probe motion. Taking advantage of the large fluorescence enhancement associated with binding of FlAsH to CaM, we determined rates of binding of FlAsH to apo-CaM and calcium-activated CaM to be 2800 +/- 80 and 310 +/- 10 M(-)(1) s(-)(1), respectively. There is no difference in the solvent accessibility of the bound FlAsH irrespective of calcium binding to CaM. Thus, given that FlAsH selectively labels disordered structures, the large difference in rates of FlAsH binding indicates that calcium binding stabilizes helix A. Frequency domain anisotropy measurements of bound FlAsH indicate that prior to calcium activation, helix A undergoes large amplitude nanosecond motions. Following calcium activation, helix A becomes immobile, and structurally coupled to the overall rotation of CaM. We discuss these results in the context of a model that suggests stabilization of helix A relative to other domain elements in the CaM structure is critical to defining high-affinity binding clefts, and in promoting specific and ordered binding of the opposing lobes of CaM to target proteins.

[Indexed for MEDLINE]

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