FROM SYNTHETIC COILED COILS TO FUNCTIONAL PROTEINS - AUTOMATED DESIGNOF A RECEPTOR FOR THE CALMODULIN-BINDING DOMAIN OF CALCINEURIN

A series of synthetic receptors capable of binding to the calmodulin-b inding domain of calcineurin (CN393-414) was designed, synthesized and characterized. The design was accomplished by docking CN393-414 again st a two-helix receptor, using an idealized three-stranded coiled coil as a starting geometry. The sequence of the receptor was chosen using a sidechain re-packing program, which employed a genetic algorithm to select potential binders from a total of 7.5 x 10(6) possible sequenc es. A total of 25 receptors were prepared, representing 13 sequences p redicted by the algorithm as well as 12 related sequences that were no t predicted. The receptors were characterized by CD spectroscopy, anal ytical ultracentrifugation, and binding assays. The receptors predicte d by the algorithm bound CN393-414 with apparent dissociation constant s ranging from 0.2 mu M to >50 mu M. Many of the receptors that were n ot predicted by the algorithm also bound to CN393-414. Methods to circ umvent this problem and to improve the automated design of functional proteins are discussed. (C) 1998 Academic Press.