Role of Flavin Mononucleotide in the Thermostability and Oligomerization of Escherichia coli Stress-Defense Protein WrbA
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- 作者：Antonino Natalello ; Silvia Maria Doglia ; Jannette Carey ; Rita Grandori
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：January 16, 2007
- 年：2007
- 卷：46
- 期：2
- 页码：543 - 553
- 全文大小：336K
- 年卷期：v.46,no.2(January 16, 2007)
- ISSN：1520-4995
文摘
WrbA is an oligomeric flavodoxin-like protein that binds one molecule of flavin mononucleotide(FMN) per monomer and whose redox activity is implicated in oxidative stress defense. WrbAthermostability and oligomerization in the presence and absence of bound FMN were investigated usingcomplementary biophysical methods. Infrared spectroscopy indicates similar structures for apo andholoWrbA. FMN binding has a dramatic effect on WrbA thermal stability, shifting the Tm by ~40 
C.Upon denaturation, the protein forms insoluble aggregates that lack native secondary structure and haveno bound FMN. Circular dichroism (CD) reveals that the thermal unfolding of apo and holoWrbA proceedsvia the formation of an aggregation-prone intermediate that retains substantial secondary structure buthas lost the native configuration of the active site. This intermediate persists in solution up to 100 C atmicromolar concentrations. A similar partially folded state is populated during chemical denaturationwith guanidinium chloride, but accumulation of the intermediate is evident only in the absence of FMN.The results also suggest that WrbA maintains some interaction with FMN in its partially folded state,despite the loss of the induced CD signal of FMN. On the basis of these data, the unfolding process canbe depicted as follows: native holoprotein 
holointermediate apointermediate insoluble aggregate.Mass spectrometry shows that FMN promotes WrbA association into tetramers, which are morethermoresistant than dimers or monomers, suggesting that multimerization underlies the FMN effect onWrbA thermostability. This study illustrates the utility of analyzing conformational transitions andintermolecular interactions using methods that probe the liquid, solid, and gas phases.
C.Upon denaturation, the protein forms insoluble aggregates that lack native secondary structure and haveno bound FMN. Circular dichroism (CD) reveals that the thermal unfolding of apo and holoWrbA proceedsvia the formation of an aggregation-prone intermediate that retains substantial secondary structure buthas lost the native configuration of the active site. This intermediate persists in solution up to 100 C atmicromolar concentrations. A similar partially folded state is populated during chemical denaturationwith guanidinium chloride, but accumulation of the intermediate is evident only in the absence of FMN.The results also suggest that WrbA maintains some interaction with FMN in its partially folded state,despite the loss of the induced CD signal of FMN. On the basis of these data, the unfolding process canbe depicted as follows: native holoprotein holointermediate apointermediate insoluble aggregate.Mass spectrometry shows that FMN promotes WrbA association into tetramers, which are morethermoresistant than dimers or monomers, suggesting that multimerization underlies the FMN effect onWrbA thermostability. This study illustrates the utility of analyzing conformational transitions andintermolecular interactions using methods that probe the liquid, solid, and gas phases.