Non-native States Research Articles

Characterization of TDP-43 RRM2 Partially Folded States and Their Significance to ALS Pathogenesis

The human protein TDP-43 is a major component of the cellular aggregates found in amyotrophic lateral sclerosis and other neurodegenerative diseases. Insoluble cytoplasmic aggregates isolated from the brain of amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients contain ubiquitinated, hyperphosphorylated, and N-terminally truncated TDP-43. Truncated fragments of TDP-43 identified from patient tissues contain part of the second RNA recognition motif (RRM2) and the disordered C-terminus, indicating that both domains can be involved in aggregation and toxicity. Here, we focus on RRM2. Using all-atom replica-averaged metadynamics simulations with NMR chemical shift restraints, we characterized the atomic structure of non-native states of RRM2, sparsely populated under native conditions. These structures reveal the exposure to the solvent of aggregation-prone peptide regions, normally buried in the native state, supporting a role in aggregation for the partially folded states of RRM2.

Specialized chaperones required

Protein Folding Although some proteins can reach a properly folded state without assistance, many require help to adopt the correct topology and avoid kinetic trapping in nonnative states. Chaperones encapsulate guest proteins and use adenosine triphosphate (ATP)–driven conformational changes to help them fold, but not all chaperones work for all substrates. Balchin et al. compared the folding pathway of the cytoskeleton protein actin with its proper chaperone, TRiC, to the incorrect folding that occurs with the bacterial chaperone GroEL. TRiC functions by stabilizing an extended form of actin with the proper secondary structure and topology. ATP binding and hydrolysis drives release of this partially folded intermediate into the chaperone where it can successfully fold. GroEL fails to bind the intermediate properly and thus is not able to successfully fold actin, even after ATP binding and hydrolysis. Cell 174 , 1507 (2018).

Peroxidase activity of cytochrome c in its compact state depends on dynamics of the heme region

Cytochrome c (cyt c) is a small globular hemoprotein with the main function as an electron carrier in mitochondrial respiratory chain. Cyt c possesses also peroxidase-like activity in the native state despite its six-coordinated heme iron. In this work, we studied the effect of increasing urea concentration in the range from 0 M to 6 M at pH 7 (pH value of the bulk solvent) and pH 5 (pH value close to negatively charged membrane) on peroxidase-like activity of cyt c. We show that peroxidase-like activity, measured by guaiacol oxidation and the ferrous oxidation in xylenol orange methods, correlates with the accessibility of the heme iron, which was assessed from the association rate constant of cyanide binding to cyt c. Cyt c peroxidase-like activity linearly increases in the pre-denaturational urea concentrations (0–4 M) at both studied pHs without an apparent formation of penta-coordinated state of the heme iron. Our results suggest that dynamic equilibrium among the denaturant-induced non-native coordination states of cyt c, very likely due to reversible unfolding of the least stable foldons, is pre-requisite for enhanced peroxidase-like activity of cyt c in its compact state. Dynamic replacement of the native sixth coordination bond of methionine-80 by lysines (72, 73, and 79) and partially also by histidines (26 and 33) provides an efficient way how to increase peroxidase-like activity of cyt c without significant conformational change at physiological conditions.

Política, policía y violencia: La regulación del Narcotráfico en el Cono Sur

Despite being a transnational organized crime, drug trafficking has a local impact in terms of security and violence, which is typically managed by non-national state actors. This paper proposes that, given their juridical and material constraints, subnational state agencies, primarily police forces, regulate drug trafficking through a combination of toleration, repression and rent extraction. I also argue that greater coordination within law enforcement agencies at the subnational level leads to lower drug-related violence at the retail dealing level. I illustrate this argument with a subnational comparison of four cases in Argentina and Brazil during the last two decades.

Compressibility of the protein-water interface.

The compressibility of a protein relates to its stability, flexibility, and hydrophobic interactions, but the measurement, interpretation, and computation of this important thermodynamic parameter present technical and conceptual challenges. Here, we present a theoretical analysis of protein compressibility and apply it to molecular dynamics simulations of four globular proteins. Using additively weighted Voronoi tessellation, we decompose the solution compressibility into contributions from the protein and its hydration shells. We find that positively cross-correlated protein-water volume fluctuations account for more than half of the protein compressibility that governs the protein's pressure response, while the self correlations correspond to small (∼0.7%) fluctuations of the protein volume. The self compressibility is nearly the same as for ice, whereas the total protein compressibility, including cross correlations, is ∼45% of the bulk-water value. Taking the inhomogeneous solvent density into account, we decompose the experimentally accessible protein partial compressibility into intrinsic, hydration, and molecular exchange contributions and show how they can be computed with good statistical accuracy despite the dominant bulk-water contribution. The exchange contribution describes how the protein solution responds to an applied pressure by redistributing water molecules from lower to higher density; it is negligibly small for native proteins, but potentially important for non-native states. Because the hydration shell is an open system, the conventional closed-system compressibility definitions yield a pseudo-compressibility. We define an intrinsic shell compressibility, unaffected by occupation number fluctuations, and show that it approaches the bulk-water value exponentially with a decay "length" of one shell, less than the bulk-water compressibility correlation length. In the first hydration shell, the intrinsic compressibility is 25%-30% lower than in bulk water, whereas its self part is 15%-20% lower. These large reductions are caused mainly by the proximity to the more rigid protein and are not a consequence of the perturbed water structure.

Does N-terminal huntingtin function as a 'holdase' for inhibiting cellular protein aggregation?

Proteolytic cleavage of huntingtin gives rise to N-terminal fragments. While the role of truncated mutant huntingtin is described in Huntington's disease (HD) pathogenesis, the function of N-terminal wild-type protein is less studied. The yeast model of HD is generated by the presence of FLAG tag and absence of polyproline tract as flanking sequences of the elongated polyglutamine stretch. We show that the same sequence derived from wild-type huntingtin exon1 is able to inhibit the aggregation of proteins in vitro and in yeast cells. It is able to stabilize client proteins as varied as luciferase, α-synuclein, and p53 in a soluble but non-native state. This is somewhat similar to the 'holdase' function of small heat shock proteins and 'nonchaperone proteins' which are able to stabilize partially unfolded client proteins in a nonspecific manner, slowing down their aggregation. Mutagenesis studies show this property to be localized at the N17 domain preceding the polyglutamine tract. Distortion of this ordered segment, either by deletion of this segment or mutation of a single residue (L4A), leads to decreased stability and increased aggregation of client proteins. It is interesting to note that the helical conformation of the N17 domain is also essential for aggregation of the N-terminal mutant protein. Our results provide evidence for a novel function for the amphipathic helix derived from exon1 of wild-type huntingtin.

P-213 - Structural and functional characterization of the acid and alkaline transition of cytochrome b5

We have described, very recently, that cytochrome b5 (Cb5) is able to acquire a peroxidase-like activity in extreme alkaline media, when a hemichromic state is attained. At pH 4, Cb5 is mainly in a high-spin state (protein unfolding occurs and heme is released from the peptide chain) and its peroxidase activity is weak when compared with the one measured to pH 12. A structural characterization of Cb5 alkaline transition allowed us to reveal several parameters that might affect its stability and activity. H1NMR studies above pH 11 indicate the existence of a heme spin alterations that correlates with formation of tyrosinate anion, suggesting conformational changes at the heme crevice. Spectroscopic data support the interaction of Cb5 with imidazole above pH 11 and suggest the appearance of a binding site in the protein that allows accessibility of substrates to the heme. Our study, accordingly with other reports, shows acquisition of enzymatic activities when non-native states are acquired, before unfolding.

Dual function of the trigger factor chaperone in nascent protein folding

Multi‐domain proteins – constituting a large group in all proteomes – often require help from molecular chaperones to fold productively, even before the ribosome has finished their synthesis. The mechanisms underlying chaperone function remain poorly understood. We have used optical tweezers to study the folding of elongation factor G (EF‐G), a model multi‐domain protein, as it emerges from the ribosome. We find that the N‐terminal G‐domain in nascent EF‐G polypeptides folds robustly. The following domain II, in contrast, fails to fold efficiently. Strikingly, interactions with the unfolded domain II convert the natively folded G domain to a non‐native state. This non‐native state readily unfolds, and the two unfolded domains subsequently form misfolded states, preventing productive folding. Both the conversion of natively folded domains and non‐productive interactions among unfolded domains are efficiently prevented by the ribosome‐binding chaperone trigger factor. Thus, our single‐molecule measurements of multi‐domain protein folding reveal an unexpected role for the chaperone: It protects already folded domains against denaturation resulting from interactions with parts of the nascent polypeptide that are not folded yet. Previous studies had implicated trigger factor in guiding the folding of individual domains, and interactions among domains had been neglected. Avoiding early folding defects is crucial, since they can propagate and result in misfolding of the entire protein. Our experiments define the folding pathway for a complex multi‐domain protein and shed light on the molecular mechanism employed by molecular chaperones to ensure productive and efficient folding.Support or Funding InformationCMK acknowledges support by the Pew Scholars Program in the Biomedical Sciences and the National Institute of General Medical Sciences (1R01GM121567)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chaperones convert the energy from ATP into the nonequilibrium stabilization of native proteins.

During and after protein translation, molecular chaperones require ATP hydrolysis to favor the native folding of their substrates and, under stress, to avoid aggregation and revert misfolding. Why do some chaperones need ATP, and what are the consequences of the energy contributed by the ATPase cycle? Here, we used biochemical assays and physical modeling to show that the bacterial chaperones GroEL (Hsp60) and DnaK (Hsp70) both use part of the energy from ATP hydrolysis to restore the native state of their substrates, even under denaturing conditions in which the native state is thermodynamically unstable. Consistently with thermodynamics, upon exhaustion of ATP, the metastable native chaperone products spontaneously revert to their equilibrium non-native states. In the presence of ATPase chaperones, some proteins may thus behave as open ATP-driven, nonequilibrium systems whose fate is only partially determined by equilibrium thermodynamics.

Neuroglobin is capable of self-oxidation of methionine64 introduced at the heme axial position.

Neuroglobin (Ngb), with its physiological role not fully understood, was found to be capable of self-oxidation of methionine64 introduced at the heme axial position (H64M Ngb), adopting a high-spin heme state and producing both methionine sulfoxide (SO-Met) and sulfone (SO2-Met), which represents the structure and function of cytochrome c in a non-native state.

Induction of a Tier-1-Like Phenotype in Diverse Tier-2 Isolates by Agents That Guide HIV-1 Env to Perturbation-Sensitive, Nonnative States.

The envelope glycoproteins (Envs) on the surfaces of HIV-1 particles are targeted by host antibodies. Primary HIV-1 isolates demonstrate different global sensitivities to antibody neutralization; tier-1 isolates are sensitive, whereas tier-2 isolates are more resistant. Single-site mutations in Env can convert tier-2 into tier-1-like viruses. We hypothesized that such global change in neutralization sensitivity results from weakening of intramolecular interactions that maintain Env integrity. Three strategies commonly applied to perturb protein structure were tested for their effects on global neutralization sensitivity: exposure to low temperature, Env-activating ligands, and a chaotropic agent. A large panel of diverse tier-2 isolates from clades B and C was analyzed. Incubation at 0°C, which globally weakens hydrophobic interactions, causes gradual and reversible exposure of the coreceptor-binding site. In the cold-induced state, Envs progress at isolate-specific rates to unstable forms that are sensitive to antibody neutralization and then gradually lose function. Agents that mimic the effects of CD4 (CD4Ms) also induce reversible structural changes to states that exhibit isolate-specific stabilities. The chaotropic agent urea (at low concentrations) does not affect the structure or function of native Env. However, urea efficiently perturbs metastable states induced by cold and CD4Ms and increases their sensitivity to antibody neutralization and their inactivation rates Therefore, chemical and physical agents can guide Env from the stable native state to perturbation-sensitive forms and modulate their stability to bestow tier-1-like properties on primary tier-2 strains. These concepts can be applied to enhance the potency of vaccine-elicited antibodies and microbicides at mucosal sites of HIV-1 transmission.IMPORTANCE An effective vaccine to prevent transmission of HIV-1 is a primary goal of the scientific and health care communities. Vaccine-elicited antibodies target the viral envelope glycoproteins (Envs) and can potentially inhibit infection. However, the potency of such antibodies is generally low. Single-site mutations in Env can enhance the global sensitivity of HIV-1 to neutralization by antibodies. We found that such a hypersensitivity phenotype can also be induced by agents that destabilize protein structure. Exposure to 0°C or low concentrations of Env-activating ligands gradually guides Env to metastable forms that expose cryptic epitopes and that are highly sensitive to neutralization. Low concentrations of the chaotropic agent urea do not affect native Env but destabilize perturbed states induced by cold or CD4Ms and increase their neutralization. The concept of enhancing antibody sensitivity by chemical agents that affect the structural stability of proteins can be applied to increase the potency of topical microbicides and vaccine-elicited antibodies.

Assembly of Huntingtin headpiece into α-helical bundles.

Protein aggregation is a hallmark of neurodegenerative disorders. In this group of brain-related disorders, a disease-specific "host" protein or fragment misfolds and adopts a metastatic, aggregate-prone conformation. Often, this misfolded conformation is structurally and thermodynamically different from its native state. Intermolecular contacts, which arise in this non-native state, promote aggregation. In this regard, understanding the molecular principles and mechanisms that lead to the formation of such a non-native state and further promote the formation of the critical nucleus for fiber growth is essential. In this study, the authors analyze the aggregation propensity of Huntingtin headpiece (httNT), which is known to facilitate the polyQ aggregation, in relation to the helix mediated aggregation mechanism proposed by the Wetzel group. The authors demonstrate that even though httNT displays a degenerate conformational spectrum on its own, interfaces of macroscopic or molecular origin can promote the α-helix conformation, eliminating all other alternatives in the conformational phase space. Our findings indicate that httNT molecules do not have a strong orientational preference for parallel or antiparallel orientation of the helices within the aggregate. However, a parallel packed bundle of helices would support the idea of increased polyglutamine concentration, to pave the way for cross-β structures.

Modern empires and nation-states

Empires and nation-states are not opposed or distinct forms of polity but closely linked forms. Pre-modern empire existed without any contrasting form of polity we might call a nation-state. Rather, they contrasted with non-national state forms such as city-states, small kingdoms and mobile, nomadic polities. These in turn were in constant interaction with any neighbouring empire or empires, perhaps becoming the core of an empire themselves, perhaps taking over all or part of an existing empire, perhaps maintaining some autonomy by virtue of remoteness or lack of attractiveness, perhaps by balancing opposed empires against each other. Empires did not have a national core, and non-empires were not national. By contrast, modern empires have always had a clearly designated nation-state core and a physically separate set of non-national peripheries. This has been crucial to ensuring that when formal empire is ended, both the imperial core and the former colonies are defined as nation-states. But ex-imperial nation-states and ex-colonial nation-states are really two kinds of states. Much contemporary confusion about the prospect for a world order of nation-states revolves round the failure to make that basic distinction.

Unravelling the Non-Native Low-Spin State of the Cytochrome c-Cardiolipin Complex: Evidence of the Formation of a His-Ligated Species Only.

The interaction between cytochrome c (Cyt c) and cardiolipin (CL) plays a vital role in the early stages of apoptosis. The binding of CL to Cyt c induces a considerable increase in its peroxidase activity that has been attributed to the partial unfolding of the protein, dissociation of the Met80 axial ligand, and formation of non-native conformers. Although the interaction between Cyt c and CL has been extensively studied, there is still no consensus regarding the conformational rearrangements of Cyt c that follow the protein-lipid interaction. To rationalize the different results and gain better insight into the Cyt c-CL interaction, we have studied the formation of the CL complex of the horse heart wild-type protein and selected mutants in which residues considered to play a key role in the interaction with CL (His26, His33, Lys72, Lys73, and Lys79) have been mutated. The analysis was conducted at both room temperature and low temperatures via ultraviolet-visible absorption, resonance Raman, and electron paramagnetic resonance spectroscopies. The trigger and the sequence of CL-induced structural variations are discussed in terms of disruption of the His26-Pro44 hydrogen bond. We unequivocally identify the sixth ligand in the partially unfolded, non-native low-spin state that Cyt c can adopt following the protein-lipid interaction, as a His ligation, ruling out the previously proposed involvement of a Lys residue or an OH- ion.

Selective Probing of Non-Native Cardiolipin-Bound Conformations of Ferricytochrome C via Ferrocyanide-Mediated Photoreduction

Upon binding to cardiolipin (CL), ferricytochrome c replaces its native Met80 ligand and ultimately gains peroxidase activity, a vital step towards its dissociation from the inner mitochondrial membrane and its subsequent initiation of apoptosis. Multiple binding studies involving ferricytochrome c and CL-containing liposomes have resulted in a variety of partially conflicting binding models, but little quantitative insight into the co-existence of native and partially unfolded conformations of ferricytochrome c has been obtained thus far. While multiple groups have put forth binding models to explain this phenomenon, further validation of these models and characterization of the involved conformational states is still outstanding. Resonance Raman (RR) spectroscopy is perfect for this task due to the high sensitivity of the heme vibrational modes to ligation, oxidation, and spin state changes of the heme iron. By utilizing ferrocyanide-mediated photoreduction, a normally undesired phenomenon that is inhibited for ferricytochrome c conformations lacking Met80 ligation, we selectively probed the non-native states of ferricytochrome c bound to CL-containing liposomes both in the presence and absence of NaCl. The thus elucidated concentrations of these non-native states at different lipid concentrations and ionic strengths were consistent with a previously reported model, where different conformations of the protein were calculated based on a global thermodynamic fit to a proposed [CL] dependent equilibrium between a native-like (prone to photoreduction) and a partially unfolded bound state for which photoreduction is inhibited. RR spectral evidence suggests a dominance of hexacoordinated low spin states even at high lipid content. Only trace evidence for the presence of a high spin state could be inferred from Raman spectra, but the presence of a charge transfer band at 625 nm in the optical spectrum might point to the presence of a quantum-mixed state.

The G41S Variant of Human Cytochrome C Enhances Apoptosis via Increased Dynamics

In addition to its well-known role as a component of the electron transport chain, mitochondrial cytochrome c can also act as a peroxidase in the early stages of the intrinsic apoptosis pathway. Ferricytochrome c contains a hexacoordinated heme iron, for which H18 and M80 are the axial ligands. Residual peroxidase activity under native conditions arises from a minor populated pentacoordinated form, in which M80 is not coordinated to the heme iron. At the onset of apoptosis, binding to cardiolipin increases the proportion of the pentacoordinated form, increasing peroxidase activity which eventually leads to apoptosis. Mutations in the human gene for cytochrome c, which result in enhanced mitochondrial apoptotic activity, cause thrombocytopenia 4, an inherited autosomal dominant thrombocytopenia, characterised by a deficiency in the number of platelets in the blood and leading to abnormal bleeding. The first such mutation to be reported was G41S. Here we use stopped-flow kinetic studies of azide binding to human ferricytochrome c, backbone amide H/D exchange and 15N-relaxation dynamics measured by NMR spectroscopy to compare the wild type and G41S forms of human cytochrome c. We show that alternative conformations are kinetically and thermodynamically more readily accessible for the G41S variant than for the wild-type protein. Residue 41 is located in the 40-57 Ω-loop, and the increased loop dynamics in the G41S variant promote the dissociation from the heme iron of the M80 ligand, revealing a direct conformational link between the loop and the axial ligand to the heme iron. Increased dissociation of M80 increases the population of a peroxidase active state, which is a key non-native conformational state in apoptosis.

Long-Range Contacts in Unfolding of Two-State Proteins.

Predicting the unfolding rates of proteins remains complicated due to the intricacy present in the unfolding pathway of proteins and further it was observed that the experimental unfolding data were less while compared to folding kinetics. The aim of our present work is to show the variation in long-range contacts observed in various sequence separation bins belonging to all-α, all-β and mixed structural classes of 52 two-state proteins. In this work linear regression technique have been used and regression equations were developed using long-range contacts observed from various sequence separation bins. Also nine topological parameters developed from the 3-D structures of proteins are related with their experimental unfolding rates and their variation in correlation coefficient is observed before and after structural classification. The present work aims to show that long-range contacts formed between residues which are sequentially far and spatially close in the 3-D structure of proteins play a crucial role in the unfolding mechanism of proteins. Also importance of long-range contacts in various experimental and theoretical studies of protein folding along with NMR studies of the unfolded non-native states of proteins have been discussed.

Autoxidation of Reduced Horse Heart Cytochrome c Catalyzed by Cardiolipin-Containing Membranes.

Visible circular dichroism, absorption, and fluorescence spectroscopy were used to probe the binding of horse heart ferrocytochrome c to anionic cardiolipin (CL) head groups on the surface of 1,1',2,2'-tetraoleoyl cardiolipin (TOCL)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (20%:80%) liposomes in an aerobic environment. We found that ferrocytochrome c undergoes a conformational transition upon binding that leads to complete oxidation of the protein at intermediate and high CL concentrations. At low lipid concentrations, the protein maintains a structure that is only slightly different from its native one, whereas an ensemble of misligated predominantly hexacoordinated low-spin states become increasingly populated at high lipid concentrations. A minor fraction of conformations with either high- or quantum-mixed-spin states were detected at a CL to protein ratio of 200 (the largest one investigated). The population of the non-native state is less pronounced than that found for cytochrome c-CL interactions initiated with oxidized cytochrome c. Under anaerobic conditions, the protein maintains its reduced state but still undergoes some conformational change upon binding to CL head groups on the liposome surface. Our data suggest that CL-containing liposomes function as catalysts by reducing the activation barrier for a Fe2+ → O2 electron transfer. Adding NaCl to the existing cytochrome-liposome mixtures under aerobic conditions inhibits protein autoxidation of ferrocytochrome c and stabilizes the reduced state of the membrane-bound protein.

Analysis of Translocation-Competent Secretory Proteins by HDX-MS.

Protein folding is an intricate and precise process in living cells. Most exported proteins evade cytoplasmic folding, become targeted to the membrane, and then trafficked into/across membranes. Their targeting and translocation-competent states are nonnatively folded. However, once they reach the appropriate cellular compartment, they can fold to their native states. The nonnative states of preproteins remain structurally poorly characterized since increased disorder, protein sizes, aggregation propensity, and the observation timescale are often limiting factors for typical structural approaches such as X-ray crystallography and NMR. Here, we present an alternative approach for the in vitro analysis of nonfolded translocation-competent protein states and their comparison with their native states. We make use of hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS), a method based on differentiated isotope exchange rates in structured vs unstructured protein states/regions, and highly dynamic vs more rigid regions. We present a complete structural characterization pipeline, starting from the preparation of the polypeptides to data analysis and interpretation. Proteolysis and mass spectrometric conditions for the analysis of the labeled proteins are discussed, followed by the analysis and interpretation of HDX-MS data. We highlight the suitability of HDX-MS for identifying short structured regions within otherwise highly flexible protein states, as illustrated by an exported protein example, experimentally tested in our lab. Finally, we discuss statistical analysis in comparative HDX-MS. The protocol is applicable to any protein and protein size, exhibiting slow or fast loss of translocation competence. It could be easily adapted to more complex assemblies, such as the interaction of chaperones with nonnative protein states.

Exploring the Levinthal limit in protein folding.

According to the thermodynamic hypothesis, the native state of proteins is uniquely defined by their amino acid sequence. On the other hand, according to Levinthal, the native state is just a local minimum of the free energy and a given amino acid sequence, in the same thermodynamic conditions, can assume many, very different structures that are as thermodynamically stable as the native state. This is the Levinthal limit explored in this work. Using computer simulations, we compare the interactions that stabilize the native state of four different proteins with those that stabilize three non-native states of each protein and find that the nature of the interactions is very similar for all such 16 conformers. Furthermore, an enhancement of the degree of fluctuation of the non-native conformers can be explained by an insufficient relaxation to their local free energy minimum. These results favor Levinthal's hypothesis that protein folding is a kinetic non-equilibrium process.