Cooperative Collapse Research Articles

Effect of environment and charge patterning on the dynamics of marginally disordered proteins.

Intrinsically disordered proteins (IDPs) and proteins with long intrinsically disordered regions (IDRs) compose a large fraction of the proteome in all domains of life. Despite an extensive body of literature on “ordinary” disordered proteins—ones with high charge to hydropathy ratio—disordered proteins with lower charge to hydropathy ratio remain underinvestigated. To what extent do such “borderline” proteins compactify or perhaps even fold under physiological conditions? In addition, how does the patterning of charged residues in the primary structure of these IDPs affect the three-dimensional structure? Here we aim to characterize three proteins with various degrees of disorder in vitro and in cell using a suite of biophysical techniques. Our results show that the chain dimensions of all three proteins are sensitive to temperature, ionic strength, and crowder concentration in vitro and inside cells, as predicted from analytical and computational models. Moreover, we observe cooperative collapse for two of the three proteins, reminiscent of folding transitions of globular proteins. We further show that charge decoration is the primary predictor of IDP chain dimensions and models that rely solely on the polymer net charge fail to capture the relative chain dimensions of the mutants of the IDPs in our study.

Understanding release kinetics and collapse proof suture retention response of curcumin loaded electrospun mats based on aliphatic polyesters and their blends

The paper aims at designing and fabrication of PLA/PCL blended suture resistant electrospun mats (EMs) encapsulating non-toxic curcumin and optimization of its release behavior, to facilitate its sustained release at the targeted areas, without complexation with any chemical and/or synthetic drug. The release of curcumin from PLA/PCL blended EMs followed a diffusion-controlled mechanism, as evident from the agreement of the experimental release data with Peppas- Korsmeyer, Higuchi, and Kopcha model. The curcumin embedded EMs have effectively rendered a release confirming to a new generalized logarithmic model. PLA/PCL blended EMs have proved to be an excellent carrier system, exhibiting enhanced cumulative curcumin release with an increase in curcumin loading. The evaluation of structural and viscoelastic properties of the fabricated EMs showed an increase in modulus and strength, along with a subsequent decrease in elongation, with an increase in curcumin content. Suture-induced cooperative collapse dynamics the EMs have been found to be a three-stage process involving stable, stable-unstable, and fast-unstable structural failure corresponding to network realignment, lateral pullout/fracture of fibers, and divergent tearing along the crack path. The viscoelastic responses showed a prominent shift in glass transition temperature (Tg) of the PCL phase indicating the development of curcumin-induced microstructural changes attributed to the H-bonding interaction with polymer segments of PLA/PCL-based EMs. Our study demonstrates, functionally efficient designing of PLA/PCL-based curcumin-loaded biodegradable EMs with sustained retention of tunable mechanical properties and hydrophobicity, irrespective of the extent of (in-vitro) curcumin release.

"Cooperative collapse" of the denatured state revealed through Clausius-Clapeyron analysis of protein denaturation phase diagrams.

Protein phase diagrams have a unique potential to identify the presence of additional thermodynamic states even when non-2-state character is not readily apparent from the experimental observables used to follow protein unfolding transitions. Two-state analysis of the von Willebrand factor A3 domain has previously revealed a discrepancy in the calorimetric enthalpy obtained from thermal unfolding transitions as compared with Gibbs-Helmholtz analysis of free energies obtained from the Linear Extrapolation Method (Tischer and Auton, Prot Sci 2013; 22(9):1147-60). We resolve this thermodynamic conundrum using a Clausius-Clapeyron analysis of the urea-temperature phase diagram that defines how and the urea m-value interconvert through the slope of cm versus T, . This relationship permits the calculation of at low temperature from m-values obtained through iso-thermal urea denaturation and high temperature m-values from obtained through iso-urea thermal denaturation. Application of this equation uncovers sigmoid transitions in both cooperativity parameters as temperature is increased. Such residual thermal cooperativity of and the m-value confirms the presence of an additional state which is verified to result from a cooperative phase transition between urea-expanded and thermally-compact denatured states. Comparison of the equilibria between expanded and compact denatured ensembles of disulfide-intact and carboxyamidated A3 domains reveals that introducing a single disulfide crosslink does not affect the presence of the additional denatured state. It does, however, make a small thermodynamically favorable free energy (∼-13 ± 1 kJ/mol) contribution to the cooperative denatured state collapse transition as temperature is raised and urea concentration is lowered. The thermodynamics of this "cooperative collapse" of the denatured state retain significant compensations between the enthalpy and entropy contributions to the overall free energy.

Observation of the Cooperative Collapse in the Spontaneous Folding Process of Cytochrome C by Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy

Observation of the Cooperative Collapse in the Spontaneous Folding Process of Cytochrome C by Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy

Buckling of Elastomeric Beams Enables Actuation of Soft Machines.

Soft, pneumatic actuators that buckle when interior pressure is less than exterior provide a new mechanism of actuation. Upon application of negative pneumatic pressure, elastic beam elements in these actuators undergo reversible, cooperative collapse, and generate a rotational motion. These actuators are inexpensive to fabricate, lightweight, easy to control, and safe to operate. They can be used in devices that manipulate objects, locomote, or interact cooperatively with humans.

Mitochondria: 3-bromopyruvate vs. mitochondria? A small molecule that attacks tumors by targeting their bioenergetic diversity

Enhanced glycolysis, the classic bioenergetic phenotype of cancer cells was described by Otto Warburg approximately 90 years ago. However, the Warburg hypothesis does not necessarily imply mitochondrial dysfunction. The alkyl-halogen, 3-bromopyruvate (3BP), would not be expected to have selective targets for cancer therapy due to its high potential reactivity toward many SH side groups. Contrary to predictions, 3BP interferes with glycolysis and oxidative phosphorylation in cancer cells without side effects in normal tissues. The mitochondrial hexokinase II has been claimed as the main target. This “Organelle in focus” article presents a historical view of the use of 3BP in biochemistry and its effects on ATP-producing pathways of cancer cells. I will discuss how the alkylated enzymes contribute to the cooperative collapse of mitochondria and apoptosis. Perspectives for targeting 3BP to bioenergetics enzymes for cancer treatment will be considered.

A Comprehensive Mechanism of Fibrin Network Formation Involving Early Branching and Delayed Single- to Double-Strand Transition from Coupled Time-Resolved X-ray/Light-Scattering Detection

The formation of a fibrin network following fibrinogen enzymatic activation is the central event in blood coagulation and has important biomedical and biotechnological implications. A non-covalent polymerization reaction between macromolecular monomers, it consists basically of two complementary processes: elongation/branching generates an interconnected 3D scaffold of relatively thin fibrils, and cooperative lateral aggregation thickens them more than 10-fold. We have studied the early stages up to the gel point by fast fibrinogen:enzyme mixing experiments using simultaneous small-angle X-ray scattering and wide-angle, multi-angle light scattering detection. The coupled evolutions of the average molecular weight, size, and cross section of the solutes during the fibrils growth phase were thus recovered. They reveal that extended structures, thinner than those predicted by the classic half-staggered, double-stranded mechanism, must quickly form. Following extensive modeling, an initial phase is proposed in which single-bonded "Y-ladder" polymers rapidly elongate before undergoing a delayed transition to the double-stranded fibrils. Consistent with the data, this alternative mechanism can intrinsically generate frequent, random branching points in each growing fibril. The model predicts that, as a consequence, some branches in these expanding "lumps" eventually interconnect, forming the pervasive 3D network. While still growing, other branches will then undergo a Ca(2+)/length-dependent cooperative collapse on the resulting network scaffolding filaments, explaining their sudden thickening, low final density, and basic mechanical properties.

Kinetic processes at the demixing transition of PNIPAM solutions

Kinetic processes, which are joined with mass transport, are studied in the vicinity of the sharp LCST-type demixing transition of semi-dilute aqueous poly(N-isopropyl acrylamide) (PNIPAM) solutions. These processes are slow as compared to the highly cooperative collapse of individual polymer chains. Purely elastic properties, that are particularly sensitive to this phase transition, are addressed depending on the temperature, space and time by Brillouin spectroscopy. Above the demixing temperature Tc, we discriminate between kinetics related to the phase separation into PNIPAM-rich and PNIPAM-poor domains and kinetics connected to the impact of gravitation on the on-going phase separation. Using shallow temperature jumps of 0.3 °C, the growth of compact PNIPAM-rich agglomerates with identical gel-like mechanical consistency is provoked independently of temperature and position within the sample above Tc. Astonishingly, the transition temperature does not vary while heating or cooling the solutions across the phase transition, although the elastic properties depend strongly on space and time during the equilibration of PNIPAM concentration gradients following the re-entrance into the low-temperature phase.

Confronting three revolutions: Western European consumer co-operatives and their divergent development, 1950–2008

This article analyses the divergent development of Western European consumer co-operatives in the period from 1950 to 2008. It asks how some consumer co-ops throughout the post-war years managed to defend and even strengthen their market share and increase their membership while others saw both market shares and membership decline or evaporate. To analyse this question the paper offers a comparative analysis of three selected consumer co-ops; one case where consumer co-ops developed positively (Norway), one case showing consumer co-operative collapse (Germany) and one case where there has been quite substantial decline but no collapse (United Kingdom). The overall argument propounded is that thesuccess or decline of these co-ops was intimately linked to how they confronted three parallel transformations in the post-war food retail market: the ‘supermarket revolution’, the ‘chain store revolution’ and the ‘consumer revolution’. The divergent ability to confront these challenges was related both to external and internal factors.

In Situ Characterization of Internal Damage in TRIP‐Steel/Mg‐PSZ Composites under Compressive Stress Using X‐Ray Computed Tomography

AbstractAn experimental setup for in situ investigations under compressive stress using laboratory X‐ray computed tomography (XCT) was developed and successfully tested. Complete deformation curves can be taken. It could be shown that XCT scans are possible during brakes of the stopped in situ experiments. In this way the deformation behavior of defined sample regions can be investigated. This kind of experiments is well suited to investigate the deformation behavior of foams and other samples which are transferable for the X‐rays used. The compression of metal matrix composite foams lead to the cooperative collapse of connected cells. We observed deformation bands arising in regions of smaller cell wall thicknesses. The deformation was dependent on size, shape, and orientation of the cells under consideration. Obviously deformation bands start at bigger cells with a small cell wall thickness and some extension perpendicular to the deformation direction. The rising of this kind of deformation bands can be explained by the dramatic change of the stress distribution in the neighbor cells after the first brake of a cell wall.

Vortex-Induced Formation of Insulin Amyloid Superstructures Probed by Time-Lapse Atomic Force Microscopy and Circular Dichroism Spectroscopy

Misfolding and aggregation of proteins are multipathway processes that result in polymorphism of amyloid fibrils. While agitation is one of the most common means of inducing structural variants of fibrils (the so-called ‘amyloid strains’), there is as yet no mechanistic explanation for this effect. In this study, time-lapse atomic force microscopy has been employed to probe insulin fibrillation upon intensive agitation. At 60 °C, the initial stages of aggregation in agitated samples are similar to those in quiescent solutions; however, in vortexed samples, an abrupt and highly cooperative collapse of early filaments occurs, yielding twisted and laterally aligned aggregates with defined chiroptical properties. In the absence of any detectable birefringence and linear dichroism, the observed strong Cotton effect is attributed to twisted chiral amyloid superstructures. Early fibrils formed in agitated samples, but transferred to quiescent conditions before the collapse event, do not form the superstructures. On the other hand, mature insulin fibrils grown in quiescent samples and later subjected to rapid vortexing transform into clumps of finely broken fibers lacking the superstructural chirality and chiroptical properties of the continuously agitated samples. A generalized mechanism of inducing structural variants of amyloid fibrils by hydrodynamic forces favoring secondary nucleation events over elongation of fibrils is put forward. We propose that competition between low-aspect-ratio and high-aspect-ratio amyloidogenic pathways driven by fluid dynamics may play an important role in promoting distinct amyloid strains.

Metal Ion Dependence of Cooperative Collapse Transitions in RNA

Positively charged counterions drive RNA molecules into compact configurations that lead to their biologically active structures. To understand how the valence and size of the cations influences the collapse transition in RNA, small-angle X-ray scattering was used to follow the decrease in the radius of gyration ( R g) of the Azoarcus and Tetrahymena ribozymes in different cations. Small, multivalent cations induced the collapse of both ribozymes more efficiently than did monovalent ions. Thus, the cooperativity of the collapse transition depends on the counterion charge density. Singular value decomposition of the scattering curves showed that folding of the smaller and more thermostable Azoarcus ribozyme is well described by two components, whereas collapse of the larger Tetrahymena ribozyme involves at least one intermediate. The ion-dependent persistence length, extracted from the distance distribution of the scattering vectors, shows that the Azoarcus ribozyme is less flexible at the midpoint of transition in low-charge-density ions than in high-charge-density ions. We conclude that the formation of sequence-specific tertiary interactions in the Azoarcus ribozyme overlaps with neutralization of the phosphate charge, while tertiary folding of the Tetrahymena ribozyme requires additional counterions. Thus, the stability of the RNA structure determines its sensitivity to the valence and size of the counterions.

UV resonance Raman determination of molecular mechanism of poly(N-isopropylacrylamide) volume phase transition.

Poly(N-isopropylacrylamide) (PNIPAM) is the premier example of a macromolecule that undergoes a hydrophobic collapse when heated above its lower critical solution temperature (LCST). Here we utilize dynamic light scattering, H-NMR, and steady-state and time-resolved UVRR measurements to determine the molecular mechanism of PNIPAM's hydrophobic collapse. Our steady-state results indicate that in the collapsed state the amide bonds of PNIPAM do not engage in interamide hydrogen bonding, but are hydrogen bonded to water molecules. At low temperatures, the amide bonds of PNIPAM are predominantly fully water hydrogen bonded, whereas, in the collapsed state one of the two normal CO hydrogen bonds is lost. The NH-water hydrogen bonding, however, remains unperturbed by the PNIPAM collapse. Our kinetic results indicate a monoexponential collapse with tau approximately 360 (+/-85) ns. The collapse rate indicates a persistence length of n approximately 10. At lengths shorter than the persistence length the polymer acts as an elastic rod, whereas at lengths longer than the persistence length the polymer backbone conformation forms a random coil. On the basis of these results, we propose the following mechanism for the PNIPAM volume phase transition. At low temperatures PNIPAM adopts an extended, water-exposed conformation that is stabilized by favorable NIPAM-water solvation shell interactions which stabilize large clusters of water molecules. As the temperature increases an increasing entropic penalty occurs for the water molecules situated at the surface of the hydrophobic isopropyl groups. A cooperative transition occurs where hydrophobic collapse minimizes the exposed hydrophobic surface area. The polymer structural change forces the amide carbonyl and N-H to invaginate and the water clusters cease to be stabilized and are expelled. In this compact state, PNIPAM forms small hydrophobic nanopockets where the (i, i + 3) isopropyl groups make hydrophobic contacts. A persistent length of n approximately 10 suggests a cooperative collapse where hydrophobic interactions between adjacent hydrophobic pockets stabilize the collapsed PNIPAM.

Domain-Specific Chaperone-Induced Expansion Is Required for β-Actin Folding: A Comparison of β-Actin Conformations upon Interactions with GroEL and Tail-less Complex Polypeptide 1 Ring Complex (TRiC)

Actin, an abundant cytosolic protein in eukaryotic cells, is dependent on the interaction with the chaperonin tail-less complex polypeptide 1 ring complex (TRiC) to fold to the native state. The prokaryotic chaperonin GroEL also binds non-native beta-actin, but is unable to guide beta-actin toward the native state. In this study we identify conformational rearrangements in beta-actin, by observing similarities and differences in the action of the two chaperonins. A cooperative collapse of beta-actin from the denatured state to an aggregation-prone intermediate is observed, and insoluble aggregates are formed in the absence of chaperonin. In the presence of GroEL, however, >90% of the aggregation-prone actin intermediate is kept in solution, which shows that the binding of non-native actin to GroEL is effective. The action of GroEL on bound flourescein-labeled beta-actin was characterized, and the structural rearrangement was compared to the case of the beta-actin-TRiC complex, employing the homo fluorescence resonance energy transfer methodology previously used [Villebeck, L., Persson, M., Luan, S.-L., Hammarström, P., Lindgren, M., and Jonsson, B.-H. (2007) Biochemistry 46 (17), 5083-93]. The results suggest that the actin structure is rearranged by a "binding-induced expansion" mechanism in both TRiC and GroEL, but that binding to TRiC, in addition, causes a large and specific separation of two subdomains in the beta-actin molecule, leading to a distinct expansion of its ATP-binding cleft. Moreover, the binding of ATP and GroES has less effect on the GroEL-bound beta-actin molecule than the ATP binding to TRiC, where it leads to a major compaction of the beta-actin molecule. It can be concluded that the specific and directed rearrangement of the beta-actin structure, seen in the natural beta-actin-TRiC system, is vital for guiding beta-actin to the native state.

Microscopic Folds and Macroscopic Jerks in Compressed Lipid Monolayers

We report hitherto unrecognized cooperative behavior in the stochastic collapse of certain compressed lipid monolayers implicated in pulmonary function. The cooperativity emerges from a statistical analysis of the collapse events captured using fluorescence microscopy and digital image analysis. The collapse events involve folding of the monolayer on a micron scale, yet each event produces a macroscopic jerk of the layer. The cooperative collapse is striking for its temporal sharpness and large spatial extent.

Sequential Collapse Folding Pathway of Staphylococcal Nuclease: Entropic Activation Barriers to Hydrophobic Collapse of the Protein Core

In this paper the sequential collapse model (SCM) is applied to reveal the folding pathway of staphylococcal nuclease. It is found that there are two energetically equivalent dominant primary contacts leading potentially to two distinct folding pathways. A third weaker contact is likely to initiate an additional less populated pathway. The findings are compared with previous theoretical and experimental results, including laboratory data suggesting that the later stages of the folding pathway of staphylococcal nuclease might be kinetically controlled. The activation barriers observed to control the intermediate stages of the folding pathway are postulated to correspond to the configurational activation barriers governing the cooperative collapse phase along each of the three predicted folding pathways.

Segmented conjugated polymers

Segmented conjugated polymers, wherein the conjugation is randomly truncated by varying lengths of non-conjugated segments, form an interesting class of polymers as they not only represent systems of varying stiffness, but also ones where the backbone can be construed as being made up of chromophores of varying excitation energies. The latter feature, especially when the chromophores are fluorescent, like in MEHPPV, makes these systems particularly interesting from the photophysics point of view. Segmented MEHPPV-x samples, wherex represents the mole fraction of conjugated segments, were prepared by a novel approach that utilizes a suitable precursor wherein selective elimination of one of the two eliminatable groups is affected; the uneliminated units serve as conjugation truncations. Control of the compositionx of the precursor therefore permits one to prepare segmented MEHPPV-x samples with varying levels of conjugation (elimination). Using fluorescence spectroscopy, we have seen that even in single isolated polymer chains, energy migration from the shorter (higher energy) chromophores to longer (lower energy) ones occurs — the extent of which depends on the level of conjugation. Further, by varying the solvent composition, it is seen that the extent of energy transfer and the formation of poorly emissive inter-chromophore excitons are greatly enhanced with increasing amounts of non-solvent. A typical S-shaped curve represents the variation of emission yields as a function of composition suggestive of a cooperative collapse of the polymer coil, reminiscent of conformational transitions seen in biological macromolecules.

Relating contact order to the rate of cooperative collapse in the sequential collapse model for protein folding pathways

In this Letter the kinetics of the cooperative collapse phase of the protein folding pathway within the sequential collapse model (SCM) is studied. The SCM predicts an approximate linear dependence between the logarithm of the rate of collapse and the contact order of the native topology of the collapsing region. This result is in general agreement with previous theoretical and experimental results for the collapse of small proteins, suggesting a similarity between the kinetics of the cooperative collapse phase of the SCM multi-state folding pathway of proteins of ∼100–150 amino acids, and the observed two-state folding transitions in small proteins.

Dissecting the effect of trifluoroethanol on ribonuclease A. Subtle structural changes detected by nonspecific proteases.

With the aim to distinguish between local and global conformational changes induced by trifluoroethanol in RNase A, spectroscopic and activity measurements in combination with proteolysis by unspecific proteases have been exploited for probing structural transitions of RNase A as a function of trifluoroethanol concentration. At > 30% (v/v) trifluoroethanol (pH 8.0; 25 degrees C), circular dichroism and fluorescence spectroscopy indicate a cooperative collapse of the tertiary structure of RNase A coinciding with the loss of its enzymatic activity. In contrast to the denaturation by guanidine hydrochloride, urea or temperature, the breakdown of the tertiary structure in trifluoroethanol is accompanied by an induction of secondary structure as detected by far-UV circular dichroism spectroscopy. Proteolysis with the nonspecific proteases subtilisin Carlsberg or proteinase K, both of which attack native RNase A at the Ala20-Ser21 peptide bond, yields refined information on conformational changes, particularly in the pretransition region. While trifluoroethanol at concentrations > 40% results in a strong increase of the rate of proteolysis and new primary cleavage sites (Tyr76-Ser77, Met79-Ser80) were identified, the rate of proteolysis at trifluoroethanol concentrations < 40% (v/v) is much smaller (up to two orders of magnitude) than that of the native RNase A. The proteolysis data point to a decreased flexibility in the surrounding of the Ala20-Ser21 peptide bond, which we attribute to subtle conformational changes of the ribonuclease A molecule. These changes, however, are too marginal to alter the overall catalytic and spectroscopic properties of ribonuclease A.

Role of Topology in the Cooperative Collapse of the Protein Core in the Sequential Collapse Model. Folding Pathway of α-Lactalbumin and Hen Lysozyme

This paper further explores the recently proposed sequential collapse model (SCM) for protein folding pathways. Two specific items are considered within the SCM: (a) the cooperative collapse phase for protein folding pathways and (b) applications of the model to suggest the folding pathways of α-lactalbumin and hen lysozyme. With regard to the first goal, it is shown that major topological rearrangements of the protein core after the hydrophobic collapse are entropically unfavorable, suggesting that the final native structure is attained sequentially through a limited number of nativelike optimization steps. It is also shown that cross-links between protein loops are entropically unfavorable. This result suggests that long proteins fold in independent folding units, with each one of them corresponding to initially forming a single loop defined by a hydrophobic contact. The second objective of this paper is to illustrate further the predictive capabilities of the SCM through its application to the folding pathways of α-lactalbumin and hen lysozyme in comparison with available experimental data and previous theoretical results. It is found that the predicted folding pathways are similar for both proteins.