PMID: 9552156Apr 29, 1998Paper

Crystal structure of calcium-independent subtilisin BPN' with restored thermal stability folded without the prodomain

Proteins
O AlmogG L Gilliland

Abstract

The three-dimensional structure of a subtilisin BPN' construct that was produced and folded without its prodomain shows the tertiary structure is nearly identical to the wild-type enzyme and not a folding intermediate. The subtilisin BPN' variant, Sbt70, was cloned and expressed in Escherichia coli without the prodomain, the 77-residue N-terminal domain that catalyzes the folding of the enzyme into its native tertiary structure. Sbt70 has the high-affinity calcium-binding loop, residues 75 to 83, deleted. Such calcium-independent forms of subtilisin BPN' refold independently while retaining high levels of activity [Bryan et al., Biochemistry, 31:4937-4945, 1992]. Sbt70 has, in addition, seven stabilizing mutations, K43N, M50F, A73L, Q206V, Y217K, N218S, Q271E, and the active site serine has been replaced with alanine to prevent autolysis. The purified Sbt70 folded spontaneously without the prodomain and crystallized at room temperature. Crystals of Sbt70 belong to space group P2(1)2(1)2(1) with unit cell parameters a = 53.5 A, b = 60.3 A, and c = 83.4 A. Comparison of the refined structure with other high-resolution structures of subtilisin BPN' establishes that the conformation of Sbt70 is essentially the same as that previous...Continue Reading

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Citations

May 15, 2013·Thyroid : Official Journal of the American Thyroid Association·Marlena GodlewskaJ Paul Banga
Jun 19, 2002·Protein Science : a Publication of the Protein Society·Csaba Hetényi, David van der Spoel
Nov 1, 2008·Current Opinion in Structural Biology·Yu-Jen Chen, Masayori Inouye
Jul 9, 2011·Protein Science : a Publication of the Protein Society·Juan E DiazGregory A Weiss
Jan 13, 2015·New Biotechnology·Ljubica VojcicUlrich Schwaneberg
May 16, 2002·The Journal of Biological Chemistry·Orna AlmogGary L Gilliland
Apr 9, 2019·Open Biology·Annabel MechelaJürgen Soll
Oct 19, 2005·The Biochemical Journal·Sergey A ShiryaevAlex Y Strongin
Jan 5, 2018·Organic & Biomolecular Chemistry·Marcel SchmidtTimo Nuijens
Jan 11, 2001·Biochimica Et Biophysica Acta·P N Bryan
Dec 12, 2002·Chemical Reviews·Philip N Bryan

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