Spontaneous Unfolding and Refolding of FNIII Domains Assayed by Thiol Exchange

Fibronectin (FN) is a large extracellular matrix (ECM) protein that is made up of

type I (FNI), type II (FNII), & type III (FNIII) domains. It assembles into an insoluble

supra-­‐‑molecular structure: the fibrillar FN matrix. FN fibrillogenesis is a cell‐‑mediated process, which is initiated when FN binds to integrins on the cell surface. The FN matrix plays an important role in cell migration, proliferation, signaling & adhesion. Despite decades of research, the FN matrix is one of the least understood supra-­‐‑molecular protein assemblies. There have been several attempts to elucidate the exact mechanism of matrix assembly resulting in significant progress in the field but it is still unclear as to what are FN-­‐‑FN interactions, the nature of these interactions and the domains of FN that

are in contact with each other. FN matrix fibrils are elastic in nature. Two models have been proposed to explain the elasticity of the fibrils. The first model: the ‘domain unfolding’ model postulates that the unraveling of FNIII domains under tension explains fibril elasticity.

The second model relies on the conformational change of FN from compact to extended to explain fibril elasticity. FN contain 15 FNIII domains, each a 7-­‐‑strand beta sandwich. Earlier work from our lab used the technique of labeling a buried Cys to study the ‘domain unfolding’ model. They used mutant FNs containing a buried Cys in a single FNIII domain and found that 6 of the 15 FNIII domains label in matrix fibrils. Domain unfolding due to tension, matrix associated conformational changes or spontaneous folding and unfolding are all possible explanation for labeling of the buried Cys. The present study also uses the technique of labeling a buried Cys to address whether it is spontaneous folding and unfolding that labels FNIII domains in cell culture. We used thiol reactive DTNB to measure the kinetics of labeling of buried Cys in eleven FN III domains over a wide range of urea concentrations (0-­‐‑9M). The kinetics data were globally fit using Mathematica. The results are equivalent to those of H-­‐‑D exchange, and

provide a comprehensive analysis of stability and unfolding/folding kinetics of each

domain. For two of the six domains spontaneous folding and unfolding is possibly the reason for labeling in cell culture. For the rest of the four domains it is probably matrix associated conformational changes or tension induced unfolding.

A long-­‐‑standing debate in the protein-­‐‑folding field is whether unfolding rate

constants or folding rate constants correlate to the stability of a protein. FNIII domains all have the same ß sandwich structure but very different stabilities and amino acid sequences. Our study analyzed the kinetics of unfolding and folding and stabilities of eleven FNIII domains and our results show that folding rate constants for FNIII domains are relatively similar and the unfolding rates vary widely and correlate to stability. FN forms a fibrillar matrix and the FN-­‐‑FN interactions during matrix fibril formation are not known. FNI 1-­‐‑9 or the N-­‐‑ terminal region is indispensible for matrix formation and its major binding partner has been shown to be FNIII 2. Earlier work from our lab, using FRET analysis showed that the interaction of FNI 1-­‐‑9 with a destabilized FNIII 2 (missing the G strand, FNIII 2ΔG) reduces the FRET efficiency. This efficiency is restored in the presence of FUD (bacterial adhesion from S. pyogenes) that has been known to interact with FNI 1-­‐‑9 via a tandem ß zipper. In the present study we

use FRET analysis and a series of deletion mutants of FNIII 2ΔG to study the shortest fragment of FNIII 2ΔG that is required to bind FNI 1-­‐‑9. Our results presented here are qualitative and show that FNIII 2ΔC’EFG is the shortest fragment required to bind FNI 1-­‐‑9. Deletion of one more strand abolishes the interaction with FNI 1-­‐‑9.

Regrettable turnover

The purpose of this paper is to present a conceptual framework to identify antecedent variables that impact on the quit/stay decisions of high value workers. In this framework, the unfolding model of voluntary turnover is enhanced by consideration of job embeddedness, and affect-driven approaches. The concept of regrettable turnover is introduced to describe acute forms of dysfunction caused when key employees quit. The regret is experienced by managers, and heavily influenced by the criteria used to judge the value of such individuals. Therefore another key aim of this paper is to build on the literature of turnover functionality to propose a more detailed set of criteria reflecting the increasingly uncertain and dynamic environments in which organisations must operate.

Job embeddedness: a multifoci theoretical extension

Integrating the expanding job embeddedness (JE) literature, in this article we advance a multifoci model of JE that is theoretically grounded in conservation of resources (COR) theory. From COR theory, we posit that employees' motivation to acquire and protect resources explains why they become embedded and how they behave once embedded. Our COR-based JE model highlights contextual antecedents that clarify how employees become embedded within different foci. Its multifoci theoretical lens also illustrates how different forms of work-focused embeddedness differentially affect work outcomes and how they interact with nonwork foci to influence those outcomes. Along with directions for further research, we further discuss theoretical and practical implications of our integrative formulation.

A signature of the T → R transition in human hemoglobin

Allosteric effects in hemoglobin arise from the equilibrium between at least two energetic states of the molecule: a tense state, T, and a relaxed state, R. The two states differ from each other in the number and energy of the interactions between hemoglobin subunits. In the T state, constraints between subunits oppose the structural changes resulting from ligand binding. In the R state, these constraints are released, thus enhancing ligand-binding affinity. In the present work, we report the presence of four sites in hemoglobin that are structurally stabilized in the R relative to the T state. These sites are Hisα103(G10) and Hisα122(H5) in each α subunit of hemoglobin. They are located at the α1β1 and α2β2 interfaces of the hemoglobin tetramer, where the histidine side chains form hydrogen bonds with specific residues from the β chains. We have measured the solvent exchange rates of side chain protons of Hisα103(G10) and Hisα122(H5) in both deoxygenated and ligated hemoglobin by NMR spectroscopy. The exchange rates were found to be higher in the deoxygenated-T than in ligated-R state. Analysis of exchange rates in terms of the local unfolding model revealed that the structural stabilization free energy at each of these two histidines is larger by ≈1.5 kcal/(mol tetramer) in the R relative to the T state. The location of these histidines at the intradimeric α1β1 and α2β2 interfaces also suggests a role for these interfaces in the allosteric equilibrium of hemoglobin.

Perceptions of organizational change: A stress and coping perspective

Few organizational change studies identify the aspects of change that are salient to individuals and that influence well-being. The authors identified three distinct change characteristics: the frequency, impact and planning of change. R. S. Lazarus and S. Folkman's (1984) cognitive phenomenological model of stress and coping was used to propose ways that these change characteristics influence individuals' appraisal of the uncertainty associated with change, and, ultimately, job satisfaction and turnover intentions. Results of a repeated cross-sectional study that collected individuals' perceptions of change one month prior to employee attitudes in consecutive years indicated that while the three change perceptions were moderately to strongly intercorrelated, the change perceptions displayed differential relationships with outcomes. Discussion focuses on the importance of systematically considering individuals' subjective experience of change.

Urea-induced unfolding of Glossoscolex paulistus hemoglobin, in oxy- and cyanomet-forms: A dissociation model

The urea effect on the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) stability was studied by analytical ultracentrifugation (AUC) and small angle X-ray scattering (SAXS). AUC data show that the sedimentation coefficient distributions curves c (S), at 1.0mol/L of urea, display a single peak at 57 S, associated to the undissociated protein. The increase in urea concentration, up to 4.0mol/L, induces the appearance of smaller species, due to oligomeric dissociation. The sedimentation coefficients and molecular masses are 9.2S and 204kDa for the dodecamer (abcd)3, 5.5S and 69kDa for the tetramer (abcd), 4.1S and 52kDa for the trimer (abc) and 2.0 S and 17kDa for the monomer d, respectively. SAXS data show initially a decrease in the I(0) values due to the oligomeric dissociation, and then, above 4.0mol/L of denaturant, for oxy-HbGp, and above 6.0mol/L for cyanomet-HbGp, an increase in the maximum dimension and gyration radius is observed, due to the unfolding process. According to AUC and SAXS data the HbGp unfolding is described by two phases: the first one, at low urea concentration, below 4.0mol/L, characterizes the oligomeric dissociation, while the second one, at higher urea concentration, is associated to the unfolding of dissociated species. Our results are complementary to a recent report based on spectroscopic observations. © 2012 Elsevier B.V.

A general two-process model describes the hydrogen exchange behavior of RNase A in unfolding conditions.

When NMR hydrogen exchange was used previously to monitor the kinetics of RNase A unfolding, some peptide NH protons were found to show EX2 exchange (detected by base catalysis) in addition to the expected EX1 exchange, whose rate is limited by the kinetic unfolding process. In earlier work, two groups showed independently that a restricted two-process model successfully fits published hydrogen exchange rates of native RNase A in the range 0-0.7 M guanidinium chloride. We find that this model predicts properties that are very different from the observed properties of the EX2 exchange reactions of RNase A in conditions where guanidine-induced unfolding takes place. The model predicts that EX2 exchange should be too fast to measure by the technique used, whereas it is readily measurable. Possible explanations for the contradiction are considered here, and we show that removing the restriction from the earlier two-process model is sufficient to resolve the contradiction; instead of specifying that exchange caused by global unfolding occurs by the EX2 mechanism, we allow it to occur by the general mechanism, which includes both the EX1 and EX2 cases. It is logical to remove this restriction because global unfolding of RNase A is known to give rise to EX1 exchange in these unfolding conditions. Resolving the contradiction makes it possible to determine whether populated unfolding intermediates contribute to the EX2 exchange, and this question is considered elsewhere. The results and simulations indicate that moderate or high denaturant concentrations readily give rise to EX1 exchange in native proteins. Earlier studies showed that hydrogen exchange in native proteins typically occurs by the EX2 mechanism but that high temperatures or pH values above 7 may give rise to EX1 exchange. High denaturant concentrations should be added to the list of variables likely to cause EX1 exchange.

Inciting the social imagination to realising the dream : An unfolding story of reform in a low-socioeconomic school

This paper presentation addresses design-based research that became a catalyst for social change among a disadvantaged school community. The aim of the longitudinal research was to protoype an evidence-based model for whole school digital and print literacy pedagogy renewal among students from low socioeconomic, Indigenous, and migrant backgrounds. Applying Anthony Gidden’s principle of the “duality of structure”, the paper presentation interprets how the collective agency of researchers and the school community began to transform the structural properties of the institution in a two-way dynamism, so that the structural properties of the school were not outside of individual action, but were implicated in its reproduction and transformation.

A semantics-based approach to querying process model repositories

As business process management technology matures, organisations acquire more and more business process models. The resulting collections can consist of hundreds, even thousands of models and their management poses real challenges. One of these challenges concerns model retrieval where support should be provided for the formulation and efficient execution of business process model queries. As queries based on only structural information cannot deal with all querying requirements in practice, there should be support for queries that require knowledge of process model semantics. In this paper we formally define a process model query language that is based on semantic relationships between tasks. This query language is independent of the particular process modelling notation used, but we will demonstrate how it can be used in the context of Petri nets by showing how the semantic relationships can be determined for these nets in such a way that state space explosion is avoided as much as possible. An experiment with three large process model repositories shows that queries expressed in our language can be evaluated efficiently.

The stretching of single poly-ubiquitin molecules: Static versus dynamic disorder in the non-exponential kinetics of chain unfolding

Static disorder has recently been implicated in the non-exponential kinetics of the unfolding of single molecules of poly-ubiquitin under a constant force Kuo, Garcia-Manyes, Li, Barel, Lu, Berne, Urbakh, Klafter, and Fernandez, Proc. Natl. Acad. Sci. U. S. A. 107, 11336 (2010)]. In the present paper, it is suggested that dynamic disorder may provide a plausible, alternative description of the experimental observations. This suggestion is made on the basis of a model in which the barrier to chain unfolding is assumed to be modulated by a control parameter r that evolves in a parabolic potential under the action of fractional Gaussian noise according to a generalized Langevin equation. The treatment of dynamic disorder within this model is pursued using Zwanzig's indirect approach to noise averaging Acc. Chem. Res. 23, 148 (1990)]. In conjunction with a self-consistent closure scheme developed by Wilemski and Fixman J. Chem. Phys. 58, 4009 (1973); ibid. 60, 866 (1974)], this approach eventually leads to an expression for the chain unfolding probability that can be made to fit the corresponding experimental data very closely. (C) 2011 American Institute of Physics.

An organizing center in a planar model of neuronal excitability

This paper studies the excitability properties of a generalized FitzHugh-Nagumo model. The model differs from the classical FitzHugh-Nagumo model in that it accounts for the effect of cooperative gating variables such as activation of calcium currents. Excitability is explored by unfolding a pitchfork bifurcation that is shown to organize five different types of excitability. In addition to the three classical types of neuronal excitability, two novel types are described and distinctly associated to the presence of cooperative variables. © 2012 Society for Industrial and Applied Mathematics.

Thermodynamic analysis of ligand-induced changes in protein thermal unfolding applied to high-throughput determination of ligand affinities with extrinsic fluorescent dyes.

The quantification of protein-ligand interactions is essential for systems biology, drug discovery, and bioengineering. Ligand-induced changes in protein thermal stability provide a general, quantifiable signature of binding and may be monitored with dyes such as Sypro Orange (SO), which increase their fluorescence emission intensities upon interaction with the unfolded protein. This method is an experimentally straightforward, economical, and high-throughput approach for observing thermal melts using commonly available real-time polymerase chain reaction instrumentation. However, quantitative analysis requires careful consideration of the dye-mediated reporting mechanism and the underlying thermodynamic model. We determine affinity constants by analysis of ligand-mediated shifts in melting-temperature midpoint values. Ligand affinity is determined in a ligand titration series from shifts in free energies of stability at a common reference temperature. Thermodynamic parameters are obtained by fitting the inverse first derivative of the experimental signal reporting on thermal denaturation with equations that incorporate linear or nonlinear baseline models. We apply these methods to fit protein melts monitored with SO that exhibit prominent nonlinear post-transition baselines. SO can perturb the equilibria on which it is reporting. We analyze cases in which the ligand binds to both the native and denatured state or to the native state only and cases in which protein:ligand stoichiometry needs to treated explicitly.

Traditional scores versus IRT estimates on forced-choice tests based on a dominance model

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Use of essential and molecular dynamics to study gamma B-crystallin unfolding after non-enzymic post-translational modifications

Essential and Molecular Dynamics (ED/MD) have been used to model the conformational changes of a protein implicated in a conformational disease-cataract, the largest cause of blindness in the world-after non-enzymic post-translational modification. Cyanate modification did not significantly alter flexibility, while the Schiff's base adduct produced a more flexible N-terminal domain, and intra-secondary structure regions, than either the cyanate adduct or the native structure. Glycation also increased linker flexibility and disrupted the charge network. A number of post-translational adducts showed structural disruption around Cys15 and increased linker flexibility; this may be important in subsequent protein aggregation. Our modelling results are in accord with experimental evidence, and show that ED/MD is a useful tool in modelling conformational changes in proteins implicated in disease processes. (C) 2003 Published by Elsevier Ltd.