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. WrbA thermostability and oligomerization in the presence and absence of bound FMN were investigated using complementary biophysical methods. Infrared spectroscopy indicates similar structures for apo and holoWrbA. FMN binding has a dramatic effect on WrbA thermal stability, shifting the T-m by similar to 40 degrees C. Upon denaturation, the protein forms insoluble aggregates that lack native secondary structure and have no bound FMN. Circular dichroism (CD) reveals that the thermal unfolding of apo and holoWrbA proceeds via the formation of an aggregation-prone intermediate that retains substantial secondary structure but has lost the native configuration of the active site. This intermediate persists in solution up to 100 degrees C at micromolar concentrations. A similar partially folded state is populated during chemical denaturation with 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 can be depicted as follows: native holoprotein -> holointermediate -> apointermediate -> insoluble aggregate. Mass spectrometry shows that FMN promotes WrbA association into tetramers, which are more thermoresistant than dimers or monomers, suggesting that multimerization underlies the FMN effect on WrbA thermostability. This study illustrates the utility of analyzing conformational transitions and intermolecular interactions using methods that probe the liquid, solid, and gas phases.
Natalello, A., Doglia, S.M., Carey, J., & Grandori, R. (2007). Role of flavin mononucleotide in the thermostability and oligomerization of Escherichia coli stress-defense protein WrbA. BIOCHEMISTRY, 46(2), 543-553.
|Citazione:||Natalello, A., Doglia, S.M., Carey, J., & Grandori, R. (2007). Role of flavin mononucleotide in the thermostability and oligomerization of Escherichia coli stress-defense protein WrbA. BIOCHEMISTRY, 46(2), 543-553.|
|Tipo:||Articolo in rivista - Articolo scientifico|
|Carattere della pubblicazione:||Scientifica|
|Titolo:||Role of flavin mononucleotide in the thermostability and oligomerization of Escherichia coli stress-defense protein WrbA|
|Autori:||Natalello, A; Doglia, SM; Carey, J; Grandori, R|
|Data di pubblicazione:||16-gen-2007|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1021/bi061769c|
|Appare nelle tipologie:||01 - Articolo su rivista|