CALCIUM-INDEPENDENT SUBTILISIN BY DESIGN

A version of subtilisin BPN' lacking the high affinity calcium site (s ite A) has been produced through genetic engineering methods, and its crystal structure refined at 1.8 A resolution. This protein and the co rresponding version containing the calcium A site are described and co mpared. The deletion of residues 75-83 was made in the context of four site-specific replacements previously shown to stabilize subtilisin. The helix that in wild type is interrupted by the calcium binding loop , is continuous in the deletion mutant, with normal geometry. A few re sidues adjacent to the loop, principally those that were involved in c alcium coordination, are repositioned and/or destabilized by the delet ion. Because refolding is greatly facilitated by the absence of the Ca -loop, this protein offers a new vehicle for analysis and dissection o f the folding reaction. This is among the largest internal changes to a protein to be described at atomic resolution.