The Flavodoxin from Helicobacter pylori: Structural Determinants of Thermostability and FMN Cofactor Binding
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- 作者：Nunilo Cremades ; Adriá ; n Velazquez-Campoy ; Ernesto Freire ; Javier Sancho
- 刊名：Biochemistry
- 出版年：2008
- 出版时间：January 15, 2008
- 年：2008
- 卷：47
- 期：2
- 页码：627 - 639
- 全文大小：518K
- 年卷期：v.47,no.2(January 15, 2008)
- ISSN：1520-4995
文摘
Flavodoxin has been recently recognized as an essential protein for a number of pathogenicbacteria including Helicobacter pylori, where it has been proposed to constitute a target for antibacterialdrug development. One way we are exploring to screen for novel inhibitory compounds is to performthermal upshift assays, for which a detailed knowledge of protein thermostability and cofactor bindingproperties is of great help. However, very little is known on the stability and ligand binding properties ofH. pylori flavodoxin, and its peculiar FMN binding site together with the variety of behaviors observedwithin the flavodoxin family preclude extrapolations. We have thus performed a detailed experimentaland computational analysis of the thermostability and cofactor binding energetics of H. pylori flavodoxin,and we have found that the thermal unfolding equilibrium is more complex that any other previouslydescribed for flavodoxins as it involves the accumulation of two distinct equilibrium intermediates.Fortunately the entire stability and binding data can be satisfactorily fitted to a model, summarized in asimple phase diagram, where the cofactor only binds to the native state. On the other hand, we show howvariability of thermal unfolding behavior within the flavodoxin family can be predicted using structure-energetics relationships implemented in the COREX algorithm. The different distribution and ranges oflocal stabilities of the Anabaena and H. pylori apoflavodoxins explain the essential experimental differencesobserved: much lower Tm1, greater resistance to global unfolding, and more pronounced cold denaturationin H. pylori. Finally, a new strategy is proposed to identify using COREX structural characteristics ofequilibrium intermediate states populated during protein unfolding.