No abstract

Electrospray-ionization mass spectrometry (ESI-MS) is widely used for protein studies. It has been shown that the extent of protein ionization under nondenaturing conditions correlates well with the solvent-accessible surface area of the tridimensional structure, for either folded monomers or multimeric complexes. The goal of this study was to test whether this relation holds for unfolded proteins as well. In order to overcome the paucity of structural data, the server ProtSA was used to model the conformational ensembles of proteins in the unfolded state and generate estimates of the average solvent accessibility. The results are analyzed along with literature data or original measurements by ESI-MS. It is found that the charge-to-surface relation holds for proteins in the unfolded state, free from solvent effects. A double-log plot is derived, in close agreement with published data for folded proteins. These results suggest that the solvent-accessible surface area is a key factor determining the extent of protein ionization by electrospray, independent of the conformational state. This conclusion helps rationalizing conformational effects in protein ESI-MS. The here reported relation can be used to predict the average solvent accessibility and, hence, the state of folding of unknown proteins from ESI-MS data.

The influence of tertiary structure on the electrospray ionization mass spectra of proteins is a well known and broadly exploited phenomenon. However, the underlying mechanism is not well understood. This paper discusses the bases and the implications of the two current hypotheses (solvent accessibility and Coulombic repulsions), pointing out the remaining open questions. Evidence reported here supports a third hypothesis, i.e. that intramolecular interactions in folded proteins play a key role in determining the observed charge-state distributions. It is proposed that native protein structures stabilize to a large extent pre-existing charges of the opposite polarity to the net charge of the ion, preventing their neutralization during the electrospray process. Thus, the higher charge states of unfolded conformations, relative to the folded structure, would not derive from a more extensive ionization of the former, but rather from a higher content of neutralizing charges in the latter. This interpretation allows several other problematic observations to be explained, including the different shapes of the spectra of folded and unfolded proteins, the discrepancies between observed and predicted gas-phase reactivity of protein ions and the apparent inconsistency of positive- and negative-ion mode results.

No abstract

Lipopolysaccharide (LPS) is a major glycolipid present in the outer membrane (OM) of Gram-negative bacteria. The peculiar permeability barrier of the OM is due to the presence of LPS at the outer leaflet of this membrane that prevents many toxic compounds from entering the cell. In Escherichia coli LPS synthesized inside the cell is first translocated over the inner membrane (IM) by the essential MsbA flippase; then, seven essential Lpt proteins located in the IM (LptBCDF), in the periplasm (LptA), and in the OM (LptDE) are responsible for LPS transport across the periplasmic space and its assembly at the cell surface. The Lpt proteins constitute a transenvelope complex spanning IM and OM that appears to operate as a single device. We show here that in vivo LptA and LptC physically interact, forming a stable complex and, based on the analysis of loss-of-function mutations in LptC, we suggest that the C-terminal region of LptC is implicated in LptA binding. Moreover, we show that defects in Lpt components of either IM or OM result in LptA degradation; thus, LptA abundance in the cell appears to be a marker of properly bridged IM and OM. Collectively, our data support the recently proposed transenvelope model for LPS transport.

Intrinsically disordered proteins (IDPs) are unable to adopt a unique 3D structure under physiological conditions and thus exist as highly dynamic conformational ensembles. IDPs are ubiquitous and widely spread in the protein realm. In the last decade, compelling experimental evidence has been gathered, pointing to the ability of IDPs and intrinsically disordered regions (IDRs) to undergo liquid–liquid phase separation (LLPS), a phenomenon driving the formation of membrane-less organelles (MLOs). These biological condensates play a critical role in the spatio-temporal organization of the cell, where they exert a multitude of key biological functions, ranging from transcriptional regulation and silencing to control of signal transduction networks. After introducing IDPs and LLPS, we herein survey available data on LLPS by IDPs/IDRs of viral origin and discuss their functional implications. We distinguish LLPS associated with viral replication and trafficking of viral components, from the LLPS-mediated interference of viruses with host cell functions. We discuss emerging evidence on the ability of plant virus proteins to interfere with the regulation of MLOs of the host and propose that bacteriophages can interfere with bacterial LLPS, as well. We conclude by discussing how LLPS could be targeted to treat phase separation-associated diseases, including viral infections.

Determining the total number of charged residues corresponding to a given value of net charge for peptides and proteins in gas phase is crucial for the interpretation of mass-spectrometry data, yet it is far from being understood. Here we show that a novel computational protocol based on force field and massive density functional calculations is able to reproduce the experimental facets of well investigated systems, such as angiotensin II, bradykinin, and tryptophan-cage. The protocol takes into account all of the possible protomers compatible with a given charge state. Our calculations predict that the low charge states are zwitterions, because the stabilization due to intramolecular hydrogen bonding and salt-bridges can compensate for the thermodynamic penalty deriving from deprotonation of acid residues. In contrast, high charge states may or may not be zwitterions because internal solvation might not compensate for the energy cost of charge separation.

The protein WrbA had been identified as an Escherichia 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 ␣/␤ 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 ϳ2 M at 25°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 M. 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.WrbA was first identified by Somerville and co-workers (1) as a protein that copurified and coimmunoprecipitated with the tryptophan (Trp) repressor, TrpR. By band-shift assay, those workers concluded that WrbA influences the binding of TrpR to its operator sequence and that WrbA alone does not bind to that DNA. On this evidence, the protein was named tryptophan (W)-repressor binding protein A. Sequence analysis and homology-based structural modeling (2) suggested that WrbA was very likely to share the ␣/␤ twisted open-sheet fold characteristic of flavodoxins. Comparisons with flavodoxins and with the protein sequence data base (3) identified WrbA as the founding member of a new class of flavodoxin-like proteins containing a well conserved insertion of 24 residues following predicted strand ␤4. Two other Escherichia coli proteins, MioC and the hypothetical protein YihB, are members of the new family but are more closely related to flavodoxins than to other family members, and both lack an insertion in this region. This insertion is itself predicted to form an additional ␣/␤ segment and is a strong candidate to be the structural element responsible for experimental reports that WrbA (Ref. 1 and this work) and its homolog in yeast, Ycp4 (4), are dimeric in solution. The modeling results also suggested that the protein presents an active site crevice that cou...