Functional analysis of the polar amino acid in TatA transmembrane helix

The twin-arginine translocation (Tat) pathway utilizes the proton-motive force (PMF) to transport folded proteins across cytoplasmic membranes in bacteria and archaea, as well as across the thylakoid membrane in plants and the inner membrane in mitochondria. In most species, the minimal components required for Tat activity consist of three subunits, TatA, TatB, and TatC. Previous studies have shown that a polar amino acid is present at the N-terminus of the TatA transmembrane helix (TMH) across many different species. In order to systematically assess the functional importance of this polar amino acid in the TatA TMH in Escherichia coli, a complete set of 19-amino-acid substitutions was examined. Unexpectedly, although being preferred overall, our experiments suggest that the polar amino acid is not necessary for a functional TatA. Hydrophobicity and helix stabilizing properties of this polar amino acid were found to be highly correlated with the Tat activity. Specifically, change in charge status of the amino acid side chain due to pH resulted in a shift in hydrophobicity, which was demonstrated to impact the Tat transport activity. Furthermore, a four-residue motif at the N-terminus of the TatA TMH was identified by sequence alignment. Using a biochemical approach, the N-terminal motif was found to be functionally significant, with evidence indicating a potential role in the preference for utilizing different PMF components. Taken together, these findings yield new insights into the functionality of TatA and its potential role in the Tat transport mechanism.

Oligomerization state of the functional bacterial twin arginine translocation (Tat) receptor complex

The twin-arginine translocation (Tat) system transports folded proteins across bacterial and plastid energy transducing membranes. Ion leaks are generally considered to be mitigated by the creation and destruction of the translocation conduit in a cargo-dependent manner, a mechanism that enables tight sealing around a wide range of cargo shapes and sizes. In contrast to the variable stoichiometry of the active translocon, the oligomerization state of the receptor complex is considered more consistently stable, but has proved stubbornly difficult to establish. Here, using a single molecule photobleaching analysis of individual inverted membrane vesicles, we demonstrate that Tat receptor complexes are tetrameric in native membranes with respect to both TatB and TatC. This establishes a maximal diameter for a resting state closed pore. A large percentage of Tat-deficient vesicles explains the typical low transport efficiencies observed. This individual reaction chamber approach will facilitate examination of the effects of stochastically distributed molecules.

S100A Family Proteins and Mesothelin in Tear Proteome Are Associated With Retinal Vein Occlusion

Tear samples were collected from 88 subjects and analyzed using absolute quantitative and comparative proteomic approach. We found a large proportion (505 proteins) of tear proteome between healthy donors and subjects with retinal vein occlusion (RVO). Comparative proteomic analysis revealed 30 proteins (p<0.05) significantly differed in their quantitative property. Among them S100A6 (3.7 fmoles/ng, p<0.001), S100A8 (0.68 fmoles/ng, p<0.001), and S100A9 (2.06 fmoles/ng, p<0.001) are the most overrepresented proteins. Mesothelin was found as tear-specific protein with significant increase (1.08 fmoles/ng versus 0.54 fmoles/ng in the control, p<0.001) in the RVO group. The selected altered proteins were combined to reconstruct the customized map of protein-protein interactions with the burden of quantitating property and the context of RVO-related association. The customized interactions map (FDR<0.01) emerged inflammation and impartment of retinal hemostasis as the main RVO-associated processes. The semantic analysis of customized map encouraged the prevalence of core biological processes encompassing dysregulation of mitochondrial organization and utilization of topologically incorrect folded proteins as a consequence of oxidative stress and inflammation caused by the retinal ischemic condition. Significantly differed proteins (S100A6, S100A8, S100A9, MSL, B2M) were applied for the ROC plotting with AUC varied from 0.772 to 0.952 suggesting their association with the CRVO.

Molecular principles of recruitment and dynamics of guest proteins in liquid droplets

Despite the continuous discovery of host and guest proteins in membraneless organelles, complex host–guest interactions hinder the understanding of the molecular grammar governing liquid–liquid phase separation. In this study, we characterized the localization and dynamic properties of guest proteins in liquid droplets using single-molecule fluorescence microscopy. Eighteen guest proteins of different sizes, structures, and oligomeric states were examined in host p53 liquid droplets. Recruitment did not significantly depend on the structural properties of the guest proteins, but was moderately correlated with their length, total charge, and number of R and Y residues. In contrast, the diffusion of disordered guest proteins was comparable to that of host p53, whereas that of folded proteins varied widely. Molecular dynamics simulations suggest that folded proteins diffuse within the voids of the liquid droplet while interacting weakly with neighboring host proteins, whereas disordered proteins adapt their structures to form tight interactions with the host proteins. Our study provides insights into the key molecular principles of the localization and dynamics of guest proteins in liquid droplets.

Influence of the TorD signal peptide chaperone on Tat-dependent protein translocation

The twin-arginine translocation (Tat) pathway transports folded proteins across energetic membranes. Numerous Tat substrates contain co-factors that are inserted before transport with the assistance of redox enzyme maturation proteins (REMPs), which bind to the signal peptide of precursor proteins. How signal peptides are transferred from a REMP to a binding site on the Tat receptor complex remains unknown. Since the signal peptide mediates both interactions, possibilities include: i) a coordinated hand-off mechanism; or ii) a diffusional search after REMP dissociation. We investigated the binding interaction between substrates containing the TorA signal peptide (spTorA) and its cognate REMP, TorD, and the effect of TorD on the in vitro transport of such substrates. We found that Escherichia coli TorD is predominantly a monomer at low micromolar concentrations (dimerization KD > 50 μM), and this monomer binds reversibly to spTorA (KD ≈ 1 μM). While TorD binds to membranes (KD ≈ 100 nM), it has no apparent affinity for Tat translocons and it inhibits binding of a precursor substrate to the membrane. TorD has a minimal effect on substrate transport by the Tat system, being mildly inhibitory at high concentrations. These data are consistent with a model in which the REMP-bound signal peptide is shielded from recognition by the Tat translocon, and spontaneous dissociation of the REMP allows the substrate to engage the Tat machinery. Thus, the REMP does not assist with targeting to the Tat translocon, but rather temporarily shields the signal peptide.

Identification of potential autoantigens in anti-CCP-positive and anti-CCP-negative rheumatoid arthritis using citrulline-specific protein arrays

The presence or absence of autoantibodies against citrullinated proteins (ACPAs) distinguishes two main groups of rheumatoid arthritis (RA) patients with different etiologies, prognoses, disease severities, and, presumably, disease pathogenesis. The heterogeneous responses of RA patients to various biologics, even among ACPA-positive patients, emphasize the need for further stratification of the patients. We used high-density protein array technology for fingerprinting of ACPA reactivity. Identification of the proteome recognized by ACPAs may be a step to stratify RA patients according to immune reactivity. Pooled plasma samples from 10 anti-CCP-negative and 15 anti-CCP-positive RA patients were assessed for ACPA content using a modified protein microarray containing 1631 different natively folded proteins citrullinated in situ by protein arginine deiminases (PADs) 2 and PAD4. IgG antibodies from anti-CCP-positive RA plasma showed high-intensity binding to 87 proteins citrullinated by PAD2 and 99 proteins citrullinated by PAD4 without binding significantly to the corresponding native proteins. Curiously, the binding of IgG antibodies in anti-CCP-negative plasma was also enhanced by PAD2- and PAD4-mediated citrullination of 29 and 26 proteins, respectively. For only four proteins, significantly more ACPA binding occurred after citrullination with PAD2 compared to citrullination with PAD4, while the opposite was true for one protein. We demonstrate that PAD2 and PAD4 are equally efficient in generating citrullinated autoantigens recognized by ACPAs. Patterns of proteins recognized by ACPAs may serve as a future diagnostic tool for further subtyping of RA patients.

Generalizable Compositional Features Influencing the Proteostatic Fates of Polar Low-Complexity Domains

Protein aggregation is associated with a growing list of human diseases. A substantial fraction of proteins in eukaryotic proteomes constitutes a proteostasis network—a collection of proteins that work together to maintain properly folded proteins. One of the overarching functions of the proteostasis network is the prevention or reversal of protein aggregation. How proteins aggregate in spite of the anti-aggregation activity of the proteostasis machinery is incompletely understood. Exposed hydrophobic patches can trigger degradation by the ubiquitin-proteasome system, a key branch of the proteostasis network. However, in a recent study, we found that model glycine (G)-rich or glutamine/asparagine (Q/N)-rich prion-like domains differ in their susceptibility to detection and degradation by this system. Here, we expand upon this work by examining whether the features controlling the degradation of our model prion-like domains generalize broadly to G-rich and Q/N-rich domains. Experimentally, native yeast G-rich domains in isolation are sensitive to the degradation-promoting effects of hydrophobic residues, whereas native Q/N-rich domains completely resist these effects and tend to aggregate instead. Bioinformatic analyses indicate that native G-rich domains from yeast and humans tend to avoid degradation-promoting features, suggesting that the proteostasis network may act as a form of selection at the molecular level that constrains the sequence space accessible to G-rich domains. However, the sensitivity or resistance of G-rich and Q/N-rich domains, respectively, was not always preserved in their native protein contexts, highlighting that proteins can evolve other sequence features to overcome the intrinsic sensitivity of some LCDs to degradation.

Subpopulations of soluble, misfolded proteins commonly bypass chaperones: How it happens at the molecular level

ABSTRACTSubpopulations of soluble, misfolded proteins can bypass chaperones within cells. The scope of this phenomenon and the lifetimes of these states have not been experimentally quantified, and how such misfolding happens at the molecular level is poorly understood. We address the first issue through a meta-analysis of the experimental literature. We find that in all quantitative protein refolding-function studies, there is always a subpopulation of soluble but misfolded and less-functional protein that does not fold in the presence of one or more chaperones. This subpopulation ranges from 8% to 50% of the soluble protein molecules in solution. Fitting the experimental time traces to a kinetic model, we find these chaperone-bypassing misfolded states take months or longer to fold and function in the presence of different chaperones. We next addressed how, at the molecular level, some misfolded proteins can evade chaperones by simulating six different proteins interacting with E. coli’s GroEL and HtpG chaperones when those proteins are in folded, unfolded, or long-lived, soluble, misfolded states. We observe that both chaperones strongly bind the unfolded state and weakly bind the folded and misfolded states to a similar degree. Thus, these chaperones cannot distinguish between the folded and long-lived misfolded states of these proteins. A structural analysis reveals the misfolded states are highly similar to the native state – having a similar size, amount of exposed hydrophobic surface area, and level of tertiary structure formation. These results demonstrate that in vitro it is common for appreciable subpopulations of proteins to remain misfolded, soluble, and evade the refolding action of chaperones for very long times. Further, these results suggest that this happens because these misfolded subpopulations are near-native and therefore interact with chaperones to a similar extent as properly folded proteins. More broadly, these results indicate a mechanism in which long-time scale changes in protein structure and function can persist in cells because some protein’s non-native states can bypass components of the proteostasis machinery.TEASERNear-native, misfolded protein conformations explain why some soluble proteins fail to refold in the presence of chaperones.

Synergistic stabilization of a double mutant in chymotrypsin inhibitor 2 from a library screen in E. coli

Most single point mutations destabilize folded proteins. Mutations that stabilize a protein typically only have a small effect and multiple mutations are often needed to substantially increase the stability. Multiple point mutations may act synergistically on the stability, and it is often not straightforward to predict their combined effect from the individual contributions. Here, we have applied an efficient in-cell assay in E. coli to select variants of the barley chymotrypsin inhibitor 2 with increased stability. We find two variants that are more than 3.8 kJ mol−1 more stable than the wild-type. In one case, the increased stability is the effect of the single substitution D55G. The other case is a double mutant, L49I/I57V, which is 5.1 kJ mol−1 more stable than the sum of the effects of the individual mutations. In addition to demonstrating the strength of our selection system for finding stabilizing mutations, our work also demonstrate how subtle conformational effects may modulate stability.

Modalities of Protein Denaturation and Nature of Denaturants

Denaturation of protein is a biological phenomenon in which a protein loses its native shape due to the breaking or disruption of weak chemical bonds and interactions which makes the protein biologically inactive. It is the process where properly folded proteins formed under physiological conditions is transformed to an unfolded protein under non-physiological conditions. The process of denaturation of proteins can occur under different physiological and chemical conditions. Denaturation can be reversible or irreversible. Denaturation mostly takes places when the protein is subjected under external elements like inorganic solutes, organic solvents, acids or bases, and by heat or irradiations. The denaturing agents or denaturants widely used in protein folding or unfolding experiments are urea and guanidinium chloride (GdmCl). In denaturation, the alpha-helix structure and beta sheets structure of the native protein are disrupted and unfolds it into any random shape. We can also say that denaturation occurs due to the disruption of bonding interactions which are responsible for secondary structure and the tertiary structure of the proteins.