An engineered disulfide cross-link accelerates the refolding rate of calcium-free subtilisin by 850-fold.
The mature form of subtilisin is an unusual example of a monomeric protein with a high kinetic barrier to folding and unfolding. Using site-directed mutagenesis of subtilisin BPN', we are attempting to determine the physical and energetic nature of the kinetic barrier. The high-affinity calcium-binding site A has been shown to create a large enthalpic barrier to unfolding. Removing the calcium-binding site A from subtilisin by deleting amino acids 75-83 greatly accelerates both unfolding and refolding reactions. Here a disulfide cross-link is introduced between residues 22 and 87 in delta 75-83 subtilisin. This was done to probe the conformational entropy of the transition state for folding. The 1.8-A X-ray structure of this mutant and the effects of the cross-link on the kinetics of unfolding and refolding are reported. Consistent with an expected loss of entropy of the unfolded protein due to the cross-link, the disulfide accelerates folding relative to the uncross-linked form. The magnitude of the acceleration of folding rate (700-850-fold at 25 degrees C) indicates that residues 22 and 87 are ordered in the transition state such that the disulfide does not affect its total entropy. Although early organization of structure around amino acids 22 and 87 greatly accelerates folding, we do not know whether the early folding of this region is a highly populated folding pathway in the absence of the cross-link. The slow step in the delta 75-83 subtilisin folding reaction may be forming initial structures capable of propagating the folding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
Center for Advanced Research in Biotechnology, Maryland Biotechnology Institute, Rockville.
S
S
Strausberg
P
P
Alexander
L
L
Wang
T
T
Gallagher
G
G
Gilliland
P
P
Bryan
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D006358:Hot Temperature; D007700:Kinetics; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013381:Subtilisins / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics
D013381:Subtilisins; D002118:Calcium