Unfolding Activity Research Articles

Documenting and analyzing pre-reflective self-consciousness underlying ongoing performance optimization in elite athletes: the theoretical and methodological approach of the course-of-experience framework.

Traditional theories of motor learning emphasize the automaticity of skillful actions. However, recent research has emphasized the role of pre-reflective self-consciousness accompanying skillful action execution. In the present paper, we present the course-of-experience framework as a means of studying elite athletes' pre-reflective self-consciousness in the unfolding activity of performance optimization. We carried out a synthetic presentation of the ontological and epistemological foundation of this framework. Then we illustrated the methodology by an in-depth analysis of two elite windsurfers' courses of experience. The analysis of global and local characteristics of the riders' courses of experience reveal (a) the meaningful activities accompanying the experience of ongoing performance optimization; (b) the multidimensionality of attentional foci and the normativity of performance self-assessment; and (c) a micro-scale phenomenological description of continuous improvement. These results highlight the fruitfulness of the course-of-experience framework to describe the experience of being absorbed in an activity of performance optimization.

Stimulation of ATP Hydrolysis by ssDNA Provides the Necessary Mechanochemical Energy for G4 Unfolding

The G-quadruplex (G4) is a distinct geometric and electrophysical structure compared to classical double-stranded DNA, and its stability can impede essential cellular processes such as replication, transcription, and translation. This study focuses on the BsPif1 helicase, revealing its ability to bind independently to both single-stranded DNA (ssDNA) and G4 structures. The unfolding activity of BsPif1 on G4 relies on the presence of a single tail chain, and the covalent continuity between the single tail chain and the G4′s main chain is necessary for efficient G4 unwinding. This suggests that ATP hydrolysis-driven ssDNA translocation exerts a pull force on G4 unwinding. Molecular dynamics simulations identified a specific region within BsPif1 that contains five crucial amino acid sites responsible for G4 binding and unwinding. A “molecular wire stripper” model is proposed to explain BsPif1′s mechanism of G4 unwinding. These findings provide a new theoretical foundation for further exploration of the G4 development mechanism in Pif1 family helicases.

The Molecular Chaperone Mechanism of the C-Terminal Domain of Large-Size Subunit Catalases.

Large-size subunit catalases (LSCs) have an additional C-terminal domain (CT) that is structurally similar to Hsp31 and DJ-1 proteins, which have molecular chaperone activity. The CT of LSCs derives from a bacterial Hsp31 protein. There are two CT dimers with inverted symmetry in LSCs, one dimer in each pole of the homotetrameric structure. We previously demonstrated the molecular chaperone activity of the CT of LSCs. Like other chaperones, LSCs are abundant proteins that are induced under stress conditions and during cell differentiation in bacteria and fungi. Here, we analyze the mechanism of the CT of LSCs as an unfolding enzyme. The dimeric form of catalase-3 (CAT-3) CT (TDC3) of Neurospora crassa presented the highest activity as compared to its monomeric form. A variant of the CAT-3 CT lacking the last 17 amino acid residues (TDC3Δ17aa), a loop containing hydrophobic and charged amino acid residues only, lost most of its unfolding activity. Substituting charged for hydrophobic residues or vice versa in this C-terminal loop diminished the molecular chaperone activity in all the mutant variants analyzed, indicating that these amino acid residues play a relevant role in its unfolding activity. These data suggest that the general unfolding mechanism of CAT-3 CT involves a dimer with an inverted symmetry, and hydrophobic and charged amino acid residues. Each tetramer has four sites of interaction with partially unfolded or misfolded proteins. LSCs preserve their catalase activity under different stress conditions and, at the same time, function as unfolding enzymes.

Serine 970 of RNA helicase MOV10 is phosphorylated and controls unfolding activity and fate of mRNAs targeted for AGO2-mediated silencing

MOV10 is an RNA helicase required for organismal development and is highly expressed in postnatal brain. MOV10 is an AGO2-associated protein that is also necessary for AGO2-mediated silencing. AGO2 is the primary effector of the miRNA pathway. MOV10 has been shown to be ubiquitinated, leading to its degradation and release from bound mRNAs, but no other posttranslational modifications with functional implications have been described. Using mass spectrometry, we show that MOV10 is phosphorylated in cells at the C-terminus, specifically at serine 970 (S970). Substitution of S970 to phospho-mimic aspartic acid (S970D) blocked unfolding of an RNA G-quadruplex, similar to when the helicase domain was mutated (K531A). In contrast, the alanine substitution (S970A) of MOV10 unfolded the model RNA G-quadruplex. To examine its role in cells, our RNA-seq analysis showed that the expression of S970D causes decreased expression of MOV10 enhanced Cross-Linking Immunoprecipitation targets compared to WT. Introduction of S970A had an intermediate effect, suggesting that S970 was protective of mRNAs. In whole-cell extracts, MOV10 and its substitutions bound AGO2 comparably; however, knockdown of AGO2 abrogated the S970D-induced mRNA degradation. Thus, MOV10 activity protects mRNA from AGO2; phosphorylation of S970 restricts this activity resulting in AGO2-mediated mRNA degradation. S970 is positioned C-terminal to the defined MOV10-AGO2 interaction site and is proximal to a disordered region thatlikely modulates AGO2 interaction with target mRNAs upon phosphorylation. In summary, we provide evidence whereby MOV10 phosphorylation facilitates AGO2 association with the 3'UTR of translating mRNAs that leads to their degradation.

Molecular architecture and oligomerization of Candida glabrata Cdc13 underpin its telomeric DNA-binding and unfolding activity.

The CST complex is a key player in telomere replication and stability, which in yeast comprises Cdc13, Stn1 and Ten1. While Stn1 and Ten1 are very well conserved across species, Cdc13 does not resemble its mammalian counterpart CTC1 either in sequence or domain organization, and Cdc13 but not CTC1 displays functions independently of the rest of CST. Whereas the structures of human CTC1 and CST have been determined, the molecular organization of Cdc13 remains poorly understood. Here, we dissect the molecular architecture of Candida glabrata Cdc13 and show how it regulates binding to telomeric sequences. Cdc13 forms dimers through the interaction between OB-fold 2 (OB2) domains. Dimerization stimulates binding of OB3 to telomeric sequences, resulting in the unfolding of ssDNA secondary structure. Once bound to DNA, Cdc13 prevents the refolding of ssDNA by mechanisms involving all domains. OB1 also oligomerizes, inducing higher-order complexes of Cdc13 in vitro. OB1 truncation disrupts these complexes, affects ssDNA unfolding and reduces telomere length in C. glabrata. Together, our results reveal the molecular organization of C. glabrata Cdc13 and how this regulates the binding and the structure of DNA, and suggest that yeast species evolved distinct architectures of Cdc13 that share some common principles.

Hepatitis C virus nonstructural protein NS3 unfolds viral G-quadruplex RNA structures

Hepatitis C virus (HCV) is a major cause of liver-related diseases and hepatocellular carcinoma. The helicase domain of one of the nonstructural proteins of HCV, NS3 (nonstructural protein 3), is essential for viral replication; however, its specific biological role is still under investigation. Here, we set out to determine the interaction between a purified recombinant full length NS3 and synthetic guanine-rich substrates that represent the conserved G-quadruplex (G4)-forming sequences in the HCV-positive and HCV-negative strands. We performed fluorescence anisotropy binding, G4 reporter duplex unwinding, and G4RNA trapping assays to determine the binding and G4 unfolding activity of NS3. Our data suggest that NS3 can unfold the conserved G4 structures present within the genome and the negative strand of HCV. Additionally, we found the activity of NS3 on a G4RNA was reduced significantly in the presence of a G4 ligand. The ability of NS3 to unfold HCV G4RNA could imply a novel biological role of the viral helicase in replication.

Moving out of the here and now: An examination of frame shifts during microteaching

Microteaching is a widely adopted instructional task in teaching methods courses. While microteaching results in promotion of self-reflection and professional development among teacher candidates, there is a relative lack of understanding of the microteaching process itself. Use of a video-recorded microteaching session as data, this conversation analytic study examines how frame shifts are used as an interactional resource to manage trouble during instruction. The analyses demonstrate that the teacher addresses student trouble displays, which lead to a halt in the progressivity of the unfolding instructional activity, in two ways: (1) the teacher shifts out of the microteaching frame to address the participants as her peers in the larger classroom context, and (2) she extends the microteaching frame to highlight the discrepancy between the current frame and actual teaching. The findings have implications for implementing microteaching in teacher education.

Making meaning with metaphor in grief therapy

Abstract Metaphors play an important role in contemporary approaches to grief therapy by helping clients (re)construe their continuing relationship with the deceased. Relevant studies have illustrated the substantive elements of metaphors (i.e. sources, targets, and mappings) in this regard, often focusing on localized bursts of intense metaphoric activity. This paper highlights the extended nature of metaphoric conceptualizations and their relationship with key meaning-making processes, following the principle of ‘correspondent analysis’ as a collaborative move between language analysts and therapy practitioners. Three specific phenomena are detailed through a mixed methods analysis of 18 motivated segments in a single session of grief therapy: (i) sporadic sources, (ii) persistent sources, and (iii) metaphor ‘chaining’ across embodied and verbal activity. Their respective links to treatment objectives and processes demonstrate how metaphor theoretic constructs dovetail with therapeutic work, and suggest future avenues for modeling unfolding metaphoric activity as a time series.

Dwell times showcase how goal structure informs preschoolers' analysis of unfolding motion patterns.

Using Hard et al.'s (2011) dwell-time paradigm, 85 preschoolers (aged 2.5-4.5; 43 female; primarily from white families) advanced at their own pace through one of three slideshows. All slideshows depicted an actor reaching toward, grasping, and retrieving a ball. However, motion patterns differed for one slideshow (straight-reach) relative to the other two (arcing-reaches), and one of the arcing-reach slideshows depicted a violation of typical goal-related motion. Preschoolers' knowledge of goal structure systematically modulated attention to event boundaries across slideshows despite surface differences, even when controlling for pixel change (an index of changes in motion). These findings showcase the value of the dwell time paradigm, and illuminate how children deploy attention as goal-related expectations shape their analysis of continuously unfolding activity.

Seesaw conformations of Npl4 in the human p97 complex and the inhibitory mechanism of a disulfiram derivative

p97, also known as valosin-containing protein (VCP) or Cdc48, plays a central role in cellular protein homeostasis. Human p97 mutations are associated with several neurodegenerative diseases. Targeting p97 and its cofactors is a strategy for cancer drug development. Despite significant structural insights into the fungal homolog Cdc48, little is known about how human p97 interacts with its cofactors. Recently, the anti-alcohol abuse drug disulfiram was found to target cancer through Npl4, a cofactor of p97, but the molecular mechanism remains elusive. Here, using single-particle cryo-electron microscopy (cryo-EM), we uncovered three Npl4 conformational states in complex with human p97 before ATP hydrolysis. The motion of Npl4 results from its zinc finger motifs interacting with the N domain of p97, which is essential for the unfolding activity of p97. In vitro and cell-based assays showed that the disulfiram derivative bis-(diethyldithiocarbamate)-copper (CuET) can bypass the copper transporter system and inhibit the function of p97 in the cytoplasm by releasing cupric ions under oxidative conditions, which disrupt the zinc finger motifs of Npl4, locking the essential conformational switch of the complex.

Biophysical and Dynamic Characterization of Fine-Tuned Binding of the Human Respiratory Syncytial Virus M2-1 Core Domain to Long RNAs.

The human respiratory syncytial virus (hRSV) M2-1 protein functions as a processivity and antitermination factor of the viral polymerase complex. Here, the first evidence that the hRSV M2-1 core domain (cdM2-1) alone has an unfolding activity for long RNAs is presented and the biophysical and dynamic characterization of the cdM2-1/RNA complex is provided. The main contact region of cdM2-1 with RNA was the α1-α2-α5-α6 helix bundle, which suffered local conformational changes and promoted the RNA unfolding activity. This activity may be triggered by base-pairing recognition. RNA molecules wrap around the whole cdM2-1, protruding their termini over the domain. The α2-α3 and α3-α4 loops of cdM2-1 were marked by an increase in picosecond internal motions upon RNA binding, even though they are not directly involved in the interaction. The results revealed that the cdM2-1/RNA complex originates from a fine-tuned binding, contributing to the unraveling interaction aspects necessary for M2-1 activity.IMPORTANCE The main outcome is the molecular description of the fine-tuned binding of the cdM2-1/RNA complex and the provision of evidence that the domain alone has unfolding activity for long RNAs. This binding mode is essential in the understanding of the function in the full-length protein. Human respiratory syncytial virus (hRSV), an orthopneumovirus, stands out for the unique role of its M2-1 protein as a transcriptional antitermination factor able to increase RNA polymerase processivity.

Multi-Sensory Perception during Palpation in Japanese Midwifery Practice

This paper demonstrates ways in which vision is integrated with other modalities of perception in a manner relevant to a temporally unfolding activity. It will do so by analyzing a single episode extracted from an abdominal palpation conducted on a pregnant woman by a midwife in Japan. The episode forms a “natural experiment” through which one can demonstrate that the integrated visual and tactile perception of an object is organized differently according to the status of the ongoing activity in which it is embedded. Data are in Japanese with English translations.

Self-guided physical training as a life unfolding activity

Self-guided physical training as a life unfolding activity

YjbH Solubility Controls Spx in Staphylococcus aureus: Implication for MazEF Toxin-Antitoxin System Regulation.

Bacterial cells respond to environmental stresses by modulating their gene expression and adjusting their proteome. In Staphylococcus aureus, selective degradation by ClpP protease eliminates damaged proteins and regulates the abundance of functional proteins such as many important stress-induced transcriptional regulators. Degradation by ClpP requires the unfolding activity of partner Clp ATPases, such as ClpX and ClpC, and assistance of substrate-specific adaptor proteins such as YjbH and TrfA. Herein, we demonstrated that YjbH is aggregated in response to growth stress stimuli, such as oxidative and antibiotic stresses. In consequence, its function as an adaptor protein is compromised. YjbH controls the degradation of the stress-induced transcriptional regulator, Spx. Aggregated YjbH cannot assist Spx degradation, which results in Spx accumulation. We discovered that depending on the stress stimulus, Spx can be soluble or insoluble, and, consequently, transcriptionally active or inactive. Therefore, Spx accumulation and solubility are key components governing activation of Spx-dependent genes. Spx positively regulates expression of a ClpCP adaptor protein TrfA. TrfA in turn is required for degradation of MazE antitoxin, the unstable component of the MazEF toxin-antitoxin system, that neutralizes the endoribonuclease activity of MazF toxin. Bacterial toxin-antitoxin systems are associated with dormancy and tolerance to antibiotics that are related to chronic and relapsing infections, and it is at present a key unresolved problem in medicine. MazF activity was linked to growth stasis, yet the precise environmental signals that trigger MazE degradation and MazF activation are poorly understood. Here we propose a model where YjbH serves as a sensor of environmental stresses for downstream regulation of MazEF activity. YjbH aggregation, soluble Spx, and TrfA, coordinately control MazE antitoxin levels and consequently MazF toxin activity. This model implies that certain stress conditions culminate in modulation of MazF activity resulting in growth stasis during in vivo infections.

What Is Novel in Hegel'sPhenomenology of Spirit

AbstractWhile it has long been commonplace to advert to thePhenomenology of Spirit's peculiar prosaic form, there has been no sustained, thematic attempt to understand the relationship between that form—as a continuous, quasi-fictional narrative—and the work's philosophical content. I argue that some of what has been felt to be outlandish about the form may be better accounted for by reading it as connected to purposeful literary decisions, decisions in turn exhibiting philosophical claims about the new mode of modern self-understanding that the argument is concerned with advancing. Extending Allen Speight's suggestions that Hegel sees literature as closely connected to his theory of agency, I argue that thePhenomenology's narrative should be understood as itself specifically and deliberately novelesque. I focus on three points that help clarify the book's form as not simply in keeping with, but as expressing aspects of its content: (1) the narrative structure of consciousness (as a unified, unfolding activity through which Hegel explores the notion of actuality), (2) the theatricalizing counterpoint between the ‘in itself’ and ‘for itself’ (as a dramatic device that Hegel connects to the social underpinnings of consciousness), and (3) the role of confession and forgiveness in the argument (as a theme that Schlegel had singled out as essential to the novel, and that Hegel repurposes both to criticize and to overcome Romanticism). I do not say that thePhenomenologyis itself a novel, but that construing some of its formal features and gestures as evoking the genre of the novel can help us to see more of what is philosophically at stake in them, and therefore in the work as a whole.

Quantifying the impact of small molecule ligands on G-quadruplex stability against Bloom helicase.

G-quadruplex (GQ) stabilizing small molecule (SM) ligands have been used to stabilize human telomeric GQ (hGQ) to inhibit telomerase activity, or non-telomeric GQs to manipulate gene expression at transcription or translation level. GQs are known to inhibit DNA replication unless destabilized by helicases, such as Bloom helicase (BLM). Even though the impact of SM ligands on thermal stability of GQs is commonly used to characterize their efficacy, how these ligands influence helicase-mediated GQ unfolding is not well understood. Three prominent SM ligands (an oxazole telomestatin derivative, pyridostatin, and PhenDC3), which thermally stabilize hGQ at different levels, were utilized in this study. How these ligands influence BLM-mediated hGQ unfolding was investigated using two independent single-molecule approaches. While the frequency of dynamic hGQ unfolding events was used as the metric in the first approach, the second approach was based on quantifying the cumulative unfolding activity as a function of time. All three SM ligands inhibited BLM activity at similar levels, 2–3 fold, in both approaches. Our observations suggest that the impact of SM ligands on GQ thermal stability is not an ideal predictor for their inhibition of helicase-mediated unfolding, which is physiologically more relevant.

The archaeal RNA chaperone TRAM0076 shapes the transcriptome and optimizes the growth of Methanococcus maripaludis.

TRAM is a conserved domain among RNA modification proteins that are widely distributed in various organisms. In Archaea, TRAM occurs frequently as a standalone protein with in vitro RNA chaperone activity; however, its biological significance and functional mechanism remain unknown. This work demonstrated that TRAM0076 is an abundant standalone TRAM protein in the genetically tractable methanoarcheaon Methanococcus maripaludis. Deletion of MMP0076, the gene encoding TRAM0076, markedly reduced the growth and altered transcription of 55% of the genome. Substitution mutations of Phe39, Phe42, Phe63, Phe65 and Arg35 in the recombinant TRAM0076 decreased the in vitro duplex RNA unfolding activity. These mutations also prevented complementation of the growth defect of the MMP0076 deletion mutant, indicating that the duplex RNA unfolding activity was essential for its physiological function. A genome-wide mapping of transcription start sites identified many 5′ untranslated regions (5′UTRs) of 20–60 nt which could be potential targets of a RNA chaperone. TRAM0076 unfolded three representative 5′UTR structures in vitro and facilitated the in vivo expression of a mCherry reporter system fused to the 5′UTRs, thus behaving like a transcription anti-terminator. Flag-tagged-TRAM0076 co-immunoprecipitated a large number of cellular RNAs, suggesting that TRAM0076 plays multiple roles in addition to unfolding incorrect RNA structures. This work demonstrates that the conserved archaeal RNA chaperone TRAM globally affects gene expression and may represent a transcriptional element in ancient life of the RNA world.

Interactions between a subset of substrate side chains and AAA+ motor pore loops determine grip during protein unfolding.

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.

Attentional profiles linked to event segmentation are robust to missing information

Everyday experience consists of rapidly unfolding sensory information that humans redescribe as discrete events. Quick and efficient redescription facilitates remembering, responding to, and learning from the ongoing sensory flux. Segmentation seems key to successful redescription: the extent to which viewers can identify boundaries between event units within continuously unfolding activities predicts both memory and action performance. However, what happens to processing when boundary content is missing? Events occurring in naturalistic situations seldom receive continuous undivided attention. As a consequence, information, including boundary content, is likely sometimes missed. In this research, we systematically explored the influence of missing information by asking participants to advance at their own pace through a series of slideshows. Some slideshows, while otherwise matched in content, contained just half of the slides present in other slideshows. Missing content sometimes occurred at boundaries. As it turned out, patterns of attention during slideshow viewing were strikingly similar across matched slideshows despite missing content, even when missing content occurred at boundaries. Moreover, to the extent that viewers compensated with increased attention, missing content did not significantly undercut event recall. These findings seem to further confirm an information optimization account of event processing: event boundaries receive heightened attention because they forecast unpredictability and thus, optimize the uptake of new information. Missing boundary content sparks little change in patterns of attentional modulation, presumably because the underlying predictability parameters of the unfolding activity itself are unchanged by missing content. Optimizing information, thus, enables event processing and recall to be impressively resilient to missing content.

Hinge-Linker Elements in the AAA+ Protein Unfoldase ClpX Mediate Intersubunit Communication, Assembly, and Mechanical Activity.

The ClpXP protease plays important roles in protein homeostasis and quality control. ClpX is a ring-shaped AAA+ homohexamer that unfolds target proteins and translocates them into the ClpP peptidase for degradation. AAA+ modules in each ClpX subunit-consisting of a large AAA+ domain, a short hinge-linker element, and a small AAA+ domain-mediate the mechanical activities of the ring hexamer. Here, we investigate the roles of these hinge-linker elements in ClpX function. Deleting one hinge-linker element in a single-chain ClpX pseudohexamer dramatically decreases unfolding and degradation activity, in part by compromising the formation of closed rings, protein-substrate binding, and ClpP binding. Covalently reclosing the broken hinge-linker interface rescues activity. Deleting one hinge-linker element from a single-chain dimer or trimer prevents assembly of stable hexamers. Mutationally disrupting a hinge-linker element preserves closed-ring assembly but reduces ATP-hydrolysis cooperativity and degradation activity. These results indicate that hinge-linker length and flexibility are optimized for efficient substrate unfolding and support a model in which the hinge-linker elements of ClpX facilitate efficient degradation both by maintaining proper ring geometry and facilitating subunit-subunit communication. This model informs our understanding of ClpX as well as the larger AAA+ family of motor proteins, which play diverse roles in converting chemical into mechanical energy in all cells.