Rita Grandori

Detecting equilibrium cytochrome c folding intermediates by electrospray ionisation mass spectrometry: Two partially folded forms populate the molten-globule state

Nanoelectrospray ionization mass spectrometry (nano‐ESI‐MS) is applied to the characterization of ferric cytochromec (cytc) conformational states under different solvent conditions. The methanol‐induced molten‐globule state in the pH range 2.6–3.0 is found to be populated by two distinct, partially folded conformers IA and IB. The more compact intermediate IB resembles that induced by glycerol in acid‐unfolded cytc. The less compact one, IA, also can be induced by destabilization of the native structure by trifluoroethanol. IA and IB can be detected, in the absence of additives, around the midpoint of the acid‐induced unfolding transition, providing direct evidence for involvement of equilibrium folding intermediates in cytc conformational transitions at low pH. This study shows that mass spectrometry can contribute to the characterization of molten‐globule states of proteins by detection of distinct, although poorly populated, conformations involved in a dynamic equilibrium.

Biochemical Characterization of WrbA, Founding Member of a New Family of Multimeric Flavodoxin-like Proteins*

The protein WrbA had been identified as anEscherichia coli stationary-phase protein that copurified and coimmunoprecipitated with the tryptophan repressor. Sequences homologous to WrbA have been reported in several species of yeast and plants. We previously showed that this new family of proteins displays low but structurally significant sequence similarity with flavodoxins and that its members are predicted to share the α/β core of the flavodoxin fold but with a short conserved insertion unique to the new family, which could account for reports that some family members may be dimeric in solution. The general sequence similarity to flavodoxins suggests that the members of the new family might bind FMN, but their wide evolutionary distribution indicates that, unlike the flavodoxins, these proteins may be ubiquitous. In this paper, we report the purification and biochemical characterization of WrbA, demonstrating that the protein binds FMN specifically and is a multimer in solution. The FMN binding constant is weaker than for many flavodoxins, being ∼2 μm at 25u2009°C in 0.1 mm sodium phosphate, pH 7.2. The protein participates in a dimer-tetramer equilibrium over a wide range of solution conditions, with a midpoint at approximately 1.4 μm. One FMN binds per monomer and has no apparent effect on the multimerization equilibrium. WrbA has no effect on the affinity or mode of DNA binding by the tryptophan repressor; thus, its physiological role remains unclear. Although many proteins with flavodoxin-like domains are known to be multimers, WrbA is apparently the first characterized case in which multimerization is associated directly with the flavodoxin-like domain itself.

Characterisation of cytochrome c unfolding by nano-electrospray-ionization time-of-fight and Fourier-transform-ion-cyclotron-resonance mass spectrometry

Protein charge-state distributions (CSDs) in electrospray ionization mass spectrometry (ESI-MS) represent a sensitive tool to probe different conformational states. We describe here the effect of trifluoroethanol (TFE) on cytochrome c equilibrium unfolding at different pH by nano-ESI-MS. While even low concentrations of TFE destabilize the protein native structure at low pH, a TFE content of 2.5%–5% is found to favor cyt c folding at pH ∼7. Furthermore, we perform a comparison of CSDs obtained by time-of-flight (ToF) and Fourier transform ion cyclotron resonance (FT-ICR) mass analyzers. To this purpose, we analyze spectra of cyt c in the presence of different kinds of denaturants. In particular, experiments with 1-propanol also suggest that by using FT-ICR-MS, as previously observed on an ESI-ToF instrument, CSDs do not appear to be controlled by the solvent surface tension as predicted by the Rayleigh-charge model. Moreover, there is general good agreement in conformational effects revealed by the different instruments under several buffer conditions. Nevertheless, the ToF instrument appears to discriminate better between unfolded and partially unfolded forms.