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PEDS Advance Access published online on May 4, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh036
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Received December 31, 2003
Revised April 13, 2004
Accepted April 13, 2004

Article

Engineering thermostability in serine protease inhibitors

Hana Im 1, Myung-Jeom Ryu 2, Myeong-Hee Yu 2*

1 Department of Molecular Biology, Sejong University, 98 Gunja-dong, Kwangjin-gu, Seoul 143-747, Korea; Functional Proteomics Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Sungbuk-gu, Seoul 136-791, Korea
2 Functional Proteomics Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Sungbuk-gu, Seoul 136-791, Korea

* To whom correspondence should be addressed. E-mail: mhyu{at}kist.re.kr.


  Abstract

Unlike most globular proteins, the native form of serine protease inhibitors (serpins) is strained. Previous studies of human {alpha}1-antitrypsin, a prototype plasma serpin, revealed that various unfavorable interactions, such as overpacking of side-chains, buried polar groups and cavities, are the structural basis of the strain. The local strain could be relieved by various stabilizing single amino acid substitutions, which appeared to remove these unfavorable interactions. To improve stability of other clinically important serpin members, here we examined whether the rules found in {alpha}1-antitrypsin studies are applicable to other serpins. Amino acid substitutions were introduced at various positions in human {alpha}1-antichymotrypsin and human antithrombin III that were equivalent to the sites of stabilizing substitutions of {alpha}1-antitrypsin. Two-thirds of the substitutions increased thermostability in all serpins tested. Mutational analysis and structural examination suggest that serpins are suboptimally folded using common structural strategies at many sites, even though some structural details can vary in individual members. Results suggest that schemes discovered with {alpha}1-antitrypsin, an easily manipulative serpin, are useful basis for engineering conformational characteristics of other clinically important serpins.

Keywords: {alpha}1-antichymotrypsin, antithrombin III, serpins, stability, strain


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