A Two--Helix Extra Domain Mediates the Halophilic Character of a Plant-Type Ferredoxin from Halophilic Archaea

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• 作者：Bianca-Lucia Marg ; Kristian Schweimer ; Heinrich Sticht ; and Dieter Oesterhelt
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：January 11, 2005
• 年：2005
• 卷：44
• 期：1
• 页码：29 - 39
• 全文大小：614K
• 年卷期：v.44,no.1(January 11, 2005)
• ISSN：1520-4995

文摘

The [2Fe-2S] ferredoxin (HsFdx) of the halophilic archaeon Halobacterium salinarum exhibitsa high degree of sequence conservation with plant-type ferredoxins except for an insertion of 30 aminoacids near its N-terminus which is extremely rich in acidic amino acids. Unfolding studies reveal thatHsFdx has an unfolding temperature of ~85 C in 4.3 M NaCl, but of only 50 C in low salinity, revealingits halophilic character. The three-dimensional structure of HsFdx was determined by NMR spectroscopy,resulting in a backbone rmsd of 0.6 Å for the diamagnetic regions of the protein. Whereas the overallstructure of HsFdx is very similar to that of the plant-type ferredoxins, two additional -helices are foundin the acidic extra domain. ¹⁵N NMR relaxation studies indicate that HsFdx is rigid, and the flexibility ofresidues is similar throughout the molecule. Monitoring protein denaturation by NMR did not revealdifferences between the core fold and the acidic domain, suggesting a cooperative unfolding of both partsof the molecule. A mutant of the HsFdx in which the acidic domain is replaced with a short loop of thenonhalophilic Anabaena ferredoxin shows a considerably changed expression pattern. The halophilic wild-type protein is readily expressed in large amounts in H. salinarum, but not in Escherichia coli, whereasthe mutant ferredoxin could only be overexpressed in E. coli. The salt concentration was also found toplay a critical role for the efficiency of cluster reconstitution: the cluster of HsFdx could be reconstituted only in a solution containing molar concentrations of NaCl, while the reconstitution of the clusterin the mutant protein proceeds efficiently in low salt. These findings suggest that the acidic domainmediates the halophilic character which is reflected in its thermostability, the exclusive expression in H.salinarum, and the ability to efficiently reconstitute the iron-sulfur cluster only at high salt concentrations.