A Reversibly Unfolding Fragment of P22 Tailspike Protein with Native Structure: The Isolated -Helix Domain
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- 作者：Stefan Miller ; Benjamin Schuler ; and Robert Seckler
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：June 23, 1998
- 年：1998
- 卷：37
- 期：25
- 页码：9160 - 9168
- 全文大小：116K
- 年卷期：v.37,no.25(June 23, 1998)
- ISSN：1520-4995
文摘
The homotrimeric tailspike endorhamnosidase of phage P22 has beenused to compare invivo and in vitro folding pathways and the influence of single aminoacid substitutions thereon. Its mainstructural motif, which contains the known folding mutation sites,consists of three large right-handedparallel 
-helices. A thermodynamic analysis of the stability oftailspike is prevented by the irreversibilityof unfolding at high temperatures or high concentrations of denaturant,probably due to interdigitation ofthe domains neighboring the -helix. We therefore expressed andisolated a tailspike fragment comprisingonly its central -helix domain (residues 109-544). As shownby equilibrium ultracentrifugation, theisolated -helix is a monomer at concentrations below 1 
M andtrimerizes reversibly at higher proteinconcentrations. Both the similarity of fluorescence and CDspectra, compared to the complete protein,and the specific binding and hydrolysis of substrate suggest anativelike structure. Moreover, ureadenaturation transitions of the -helix domain are freely reversible,providing the basis for a futurequantitative analysis of the effects of the folding mutations on thethermodynamic stability of the domainand of structural features responsible for folding and stability of theparallel -helix motif in general.
-helices. A thermodynamic analysis of the stability oftailspike is prevented by the irreversibilityof unfolding at high temperatures or high concentrations of denaturant,probably due to interdigitation ofthe domains neighboring the -helix. We therefore expressed andisolated a tailspike fragment comprisingonly its central -helix domain (residues 109-544). As shownby equilibrium ultracentrifugation, theisolated -helix is a monomer at concentrations below 1 M andtrimerizes reversibly at higher proteinconcentrations. Both the similarity of fluorescence and CDspectra, compared to the complete protein,and the specific binding and hydrolysis of substrate suggest anativelike structure. Moreover, ureadenaturation transitions of the -helix domain are freely reversible,providing the basis for a futurequantitative analysis of the effects of the folding mutations on thethermodynamic stability of the domainand of structural features responsible for folding and stability of theparallel -helix motif in general.