Calcium-independent subtilisin by design.
A version of subtilisin BPN' lacking the high affinity calcium site (site A) has been produced through genetic engineering methods, and its crystal structure refined at 1.8 A resolution. This protein and the corresponding version containing the calcium A site are described and compared. 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 residues adjacent to the loop, principally those that were involved in calcium coordination, are repositioned and/or destabilized by the deletion. Because refolding is greatly facilitated by the absence of the Ca-loop, this protein offers a new vehicle for analysis and dissection of the folding reaction. This is among the largest internal changes to a protein to be described at atomic resolution.
Gallagher
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T
Center for Advanced Research in Biotechnology, Maryland Biotechnology Institute, University of Maryland, Shady Grove 20850.
Bryan
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P
Gilliland
G L
GL
D000595:Amino Acid Sequence; D002118:Calcium / Q000737:chemistry*; D005818:Genetic Engineering; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013381:Subtilisins / Q000737:chemistry* / Q000235:genetics; D014961:X-Ray Diffraction
D013381:Subtilisins; D002118:Calcium