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Commensal and pathogenic bacteria must deal with many different stress conditions to survive in and colonize the human gastrointestinal tract. One major challenge that bacteria encounter in the gut is the high concentration of bile salts, which not only aid in food absorption but also act as effective physiological antimicrobials. The mechanism by which bile salts limit bacterial growth is still largely unknown. Here, we show that bile salts cause widespread protein unfolding and aggregation, affecting many essential proteins. Simultaneously, the bacterial cytosol becomes highly oxidizing, indicative of disulfide stress. Strains defective in reducing oxidative thiol modifications, restoring redox homeostasis, or preventing irreversible protein aggregation under disulfide stress conditions are sensitive to bile salt treatment. Surprisingly, cholate and deoxycholate, two of the most abundant and very closely related physiological bile salts, vary substantially in their destabilizing effects on proteins in vitro and cause protein unfolding of different subsets of proteins in vivo. Our results provide a potential mechanistic explanation for the antimicrobial effects of bile salts, help explain the beneficial effects of bile salt mixtures, and suggest that we have identified a physiological source of protein-unfolding disulfide stress conditions in bacteria.

Dry molten globular (DMG) intermediates, an expanded form of the native protein with a dry core, have been observed during denaturant-induced unfolding of many proteins. These observations are counterintuitive because traditional models of chemical denaturation rely on changes in solvent-accessible surface area, and there is no notable change in solvent-accessible surface area during the formation of the DMG. Here we show, using multisite fluorescence resonance energy transfer, far-UV CD, and kinetic thiol-labeling experiments, that the guanidinium chloride (GdmCl)-induced unfolding of RNase H also begins with the formation of the DMG. Population of the DMG occurs within the 5-ms dead time of our measurements. We observe that the size and/or population of the DMG is linearly dependent on [GdmCl], although not as strongly as the second and major step of unfolding, which is accompanied by core solvation and global unfolding. This rapid GdmCl-dependent population of the DMG indicates that GdmCl can interact with the protein before disrupting the hydrophobic core. These results imply that the effect of chemical denaturants cannot be interpreted solely as a disruption of the hydrophobic effect and strongly support recent computational studies, which hypothesize that chemical denaturants first interact directly with the protein surface before completely unfolding the protein in the second step (direct interaction mechanism).

Chaperonins are cage-like complexes in which nonnative polypeptides prone to aggregation are thought to reach their native state optimally. However, they also may use ATP to unfold stably bound misfolded polypeptides and mediate the out-of-cage native refolding of large proteins. Here, we show that even without ATP and GroES, both GroEL and the eukaryotic chaperonin containing t-complex polypeptide 1 (CCT/TRiC) can unfold stable misfolded polypeptide conformers and readily release them from the access ways to the cage. Reconciling earlier disparate experimental observations to ours, we present a comprehensive model whereby following unfolding on the upper cavity, in-cage confinement is not needed for the released intermediates to slowly reach their native state in solution. As over-sticky intermediates occasionally stall the catalytic unfoldase sites, GroES mobile loops and ATP are necessary to dissociate the inhibitory species and regenerate the unfolding activity. Thus, chaperonin rings are not obligate confining antiaggregation cages. They are polypeptide unfoldases that can iteratively convert stable off-pathway conformers into functional proteins.

Acyldepsipeptide (ADEP) antibiotics effectively kill multidrug-resistant Gram-positive pathogens, including vancomycin-resistant enterococcus, penicillin-resistant Streptococcus pneumoniae (PRSP), and methicillin-resistant Staphylococcus aureus (MRSA). The antibacterial activity of ADEP depends on a new mechanism of action, i.e., the deregulation of bacterial protease ClpP that leads to bacterial self-digestion. Our data allow new insights into the mode of ADEP action by providing a molecular explanation for the distinct bacterial phenotypes observed at low versus high ADEP concentrations. In addition, we show that ClpP alone, in the absence of any unfoldase or energy-consuming system, and only activated by the small molecule antibiotic ADEP, leads to the unfolding of the cell division protein FtsZ. Antibiotic acyldepsipeptides (ADEPs) deregulate ClpP, the proteolytic core of the bacterial Clp protease, thereby inhibiting its native functions and concomitantly activating it for uncontrolled proteolysis of nonnative substrates. Importantly, although ADEP-activated ClpP is assumed to target multiple polypeptide and protein substrates in the bacterial cell, not all proteins seem equally susceptible. In Bacillus subtilis , the cell division protein FtsZ emerged to be particularly sensitive to degradation by ADEP-activated ClpP at low inhibitory ADEP concentrations. In fact, FtsZ is the only bacterial protein that has been confirmed to be degraded in vitro as well as within bacterial cells so far. However, the molecular reason for this preferred degradation remained elusive. Here, we report the unexpected finding that ADEP-activated ClpP alone, in the absence of any Clp-ATPase, leads to an unfolding and subsequent degradation of the N-terminal domain of FtsZ, which can be prevented by the stabilization of the FtsZ fold via nucleotide binding. At elevated antibiotic concentrations, importantly, the C terminus of FtsZ is notably targeted for degradation in addition to the N terminus. Our results show that different target structures are more or less accessible to ClpP, depending on the ADEP level present. Moreover, our data assign a Clp-ATPase-independent protein unfolding capability to the ClpP core of the bacterial Clp protease and suggest that the protein fold of FtsZ may be more flexible than previously anticipated. IMPORTANCE Acyldepsipeptide (ADEP) antibiotics effectively kill multidrug-resistant Gram-positive pathogens, including vancomycin-resistant enterococcus, penicillin-resistant Streptococcus pneumoniae (PRSP), and methicillin-resistant Staphylococcus aureus (MRSA). The antibacterial activity of ADEP depends on a new mechanism of action, i.e., the deregulation of bacterial protease ClpP that leads to bacterial self-digestion. Our data allow new insights into the mode of ADEP action by providing a molecular explanation for the distinct bacterial phenotypes observed at low versus high ADEP concentrations. In addition, we show that ClpP alone, in the absence of any unfoldase or energy-consuming system, and only activated by the small molecule antibiotic ADEP, leads to the unfolding of the cell division protein FtsZ.

Wheat α-amylase, a multi-domain protein with immense industrial applications, belongs to α+β class of proteins with native molecular mass of 32 kDa. In the present study, the pathways leading to denaturation and the relevant unfolded states of this multi-domain, robust enzyme from wheat were discerned under the influence of temperature, pH and chemical denaturants. The structural and functional aspects along with thermodynamic parameters for α-amylase unfolding were probed and analyzed using fluorescence, circular dichroism and enzyme assay methods. The enzyme exhibited remarkable stability up to 70°C with tendency to aggregate at higher temperature. Acid induced unfolding was also incomplete with respect to the structural content of the enzyme. Strong ANS binding at pH 2.0 suggested the existence of a partially unfolded intermediate state. The enzyme was structurally and functionally stable in the pH range 4.0-9.0 with 88% recovery of hydrolytic activity. Careful examination of biophysical properties of intermediate states populated in urea and GdHCl induced denaturation suggests that α-amylase unfolding undergoes irreversible and non-coincidental cooperative transitions, as opposed to previous reports of two-state unfolding. Our investigation highlights several structural features of the enzyme in relation to its catalytic activity. Since, α-amylase has been comprehensively exploited for use in a range of starch-based industries, in addition to its physiological significance in plants and animals, knowledge regarding its stability and folding aspects will promote its biotechnological applications.

Detailed understanding of protein function and malfunction hinges on the ability to characterize transiently populated states and the transitions between them. Here, we use ¹⁵N, ¹HN, and ¹³CO NMR R₂ relaxation dispersion to investigate spontaneous unfolding and refolding events of native apomyoglobin. Above pH 5.0, dispersion is dominated by processes involving fluctuations of the F-helix region, which is invisible in NMR spectra. Measurements of R₂ dispersion for residues contacted by the F-helix region in the native (N) structure reveal a transient state formed by local unfolding of helix F and undocking from the protein core. A similar state was detected at pH 4.75–4.95 and determined to be an onpathway intermediate (I1) in a linear three-state unfolding scheme (N ⇆ I1 ⇆ MG) leading to a transiently populated molten globule (MG) state. The slowest steps in unfolding and refolding are N → I1 (36 s⁻¹) and MG → I1 (26 s⁻¹), respectively. Differences in chemical shift between N and I1 are very small, except in regions adjacent to helix F, showing that their core structures are similar. Chemical shift changes between the N and MG states, obtained from R₂ dispersion, reveal that the transient MG state is structurally similar to the equilibrium MG observed previously at high temperature and low pH. Analysis of MG state chemical shifts shows the location of residual helical structure in the transient intermediate and identifies regions that unfold or rearrange into nonnative structure during the N → MG transition. The experiments also identify regions of energetic frustration that \"crack\" during unfolding and impede the refolding process.

Aging is characterized by the accumulation of dysfunctional mitochondria. Since these organelles are involved in many important cellular processes, different mechanisms exist to maintain their integrity. Among them is the mitochondrial unfolding protein response, which triggers the expression of a set of proteins aimed at re-establishing mitochondrial homeostasis. The induction of mitochondrial chaperones expression, particularly of Hsp60 and Hsp70, is a hallmark of this pathway. In Drosophila melanogaster, Hsp22 is also up-regulated by mitochondrial stress. This small heat shock protein is one of the members of the family to be localized inside mitochondria. One characteristic of Drosophila Hsp22 is its preferential up-regulation during aging and in oxidative stress conditions. It is a beneficial protein since its over-expression increases lifespan and resistance to stress while its down-regulation is detrimental. This review focuses on Drosophila Hsp22 and its links with the mitochondrial unfolding protein response and the aging process, in addition to highlight the important role of this sHSP in mitochondrial homeostasis.

Purpose To enable aggregation rate prediction over a broad temperature range for complex multi-domain proteins at high concentrations. Methods Thermal unfolding, non-isothermal kinetics and storage stability studies were conducted on a model multi-domain protein (MDP) at moderate to high concentrations (25–125 mg/mL) over a broad temperature range (4–40°C). Results Storage stability studies indicated the aggregation of MDP in solution to be a second order process. Application of Arrhenius kinetics to accelerated stability data resulted in underestimation of the aggregation rate under refrigerated conditions. Additional studies undertaken to understand the mechanism of the aggregation process highlighted the association of the monomer (or the aggregation competent species) to be the rate-limiting step for aggregation over the temperature range studied. Thermal unfolding studies in the presence of urea were used to calculate the heat capacity change upon unfolding (Δc p,un ). The resulting value of Δc p,un when used in the extended Lumry-Eyring model resulted in a more accurate and a conservative estimate of the aggregation rate under refrigerated condition. Some complicating factors for the aggregation rate prediction for multi-domain proteins at high concentration are discussed. Conclusions The work highlights (i) the significance of incorporating unfolding thermodynamics in protein aggregation rate prediction, (ii) the advantages and challenges associated with the use of the extended Lumry-Eyring (ELE) model for rate prediction and (iii) the utility of using the Arrhenius and the ELE models in tandem during product development.

Most AAA+ remodeling motors denature proteins by pulling on the peptide termini of folded substrates, but it is not well-understood how motors produce grip when resisting a folded domain. Here, at single amino-acid resolution, we identify the determinants of grip by measuring how substrate tail sequences alter the unfolding activity of the unfoldase-protease ClpXP. The seven amino acids abutting a stable substrate domain are key, with residues 2–6 forming a core that contributes most significantly to grip. ClpX grips large hydrophobic and aromatic side chains strongly and small, polar, or charged side chains weakly. Multiple side chains interact with pore loops synergistically to strengthen grip. In combination with recent structures, our results support a mechanism in which unfolding grip is primarily mediated by non-specific van der Waal’s interactions between core side chains of the substrate tail and a subset of YVG loops at the top of the ClpX axial pore. Proteins are the workhorses of the body, fulfilling many roles essential for life processes. These molecules are made up of hundreds or thousands of small units called amino acids, which attach to each other to form a long chain. The exact sequence of amino acids determines how the protein will then fold to acquire its final, three-dimentional shape. Enzymes called proteases can degrade unneeded or faulty proteins so that the amino acids can be recycled. For instance, in bacteria, the AAA+ protease ClpXP can recognize and ‘grab’ specific patterns of amino acids at the ends of a protein. This molecular machine then tugs on the segment and unfold the protein, the way a ball of yarn unwinds when pulled from one end. The unfurled protein is then fed into a different section of ClpXP, where it is chopped into short segments for recycling. ClpXP is the best-characterized enzyme amongst AAA+ proteases. However, it is still unclear how it can grip target proteins tightly enough to allow unfolding. To investigate, Bell et al. attached different patterns of 12 amino acids to the end of a folded protein. How well ClpXP grasped each of these proteins was then measured in bacteria and in test tubes. This revealed that ClpXP attaches to six to eight amino acids at a time, suggesting that only part of the enzyme clasps on the protein. Large amino acids are better gripped than small amino acids, similar to how a knotted string is easier to hold than a smooth rope. Amino acids that are electrically charged also interfere with ClpXP attaching to the protein. Finally, ClpXP grasps multiple amino acids at the same time, which dramatically increases grip strength. Many proteins, including some found in viruses, use ‘slippery’ patterns of amino acids to avoid being gripped and unfolded by proteases. By understanding how different patterns of amino acids are grasped, it may someday be possible to engineer enzymes able to target dangerous proteins.

Sulfonamide derivatives, such as sulfadiazine (SD) are used as antibiotics and, very recently, anti-amyloid properties of these have been reported. We have evaluated binding of SD with bovine serum albumin (BSA) followed by unfolding of protein. Studies were accomplished at physiological conditions of temperature (37 °C) and pH (7.4), employing UV, fluorescence, circular dichroism (CD) and Fourier transform infra-red (FTIR) spectroscopies. In presence of drug, UV spectrum of BSA was altered from the spectrum of native BSA due to the interaction between albumin and drug. Excitation of protein at 295 nm showed that fluorescence quenching of BSA by SD is a result of the formation of SD–BSA complex. The data were analyzed using Stern–Volmer and Lineweaver–Burk methods. From both methods it was evaluated that the quenching involved in BSA–SD binding was static. BSA had only one binding site for SD. Synchronous fluorescence spectra have shown a red shift and advocated that hydrophobicity around both Trp and Tyr residues was decreased. CD results revealed that the conformation of macromolecule remain undisturbed at low concentrations (up to 20 μM of the SD) and there was small perturbation in the secondary structure from 20 to 50 μM of SD followed by a large change and consequent unfolding on further increase in the drug concentration. Both synchronous and CD measurements were consistent to each other. FTIR spectra revealed the shifting of amide I band which is also an indication of conformational change of the protein. Graphical Abstract