Biophysical characterization of two different stable misfolded monomeric polypeptides that are chaperone-amenable substrates.

Misfolded polypeptide monomers may be regarded as the initial species of many protein aggregation pathways, which could accordingly serve as primary targets for molecular chaperones. It is therefore of paramount importance to study the cellular mechanisms that can prevent misfolded monomers from entering the toxic aggregation pathway and moreover rehabilitate them into active proteins. Here, we produced two stable misfolded monomers of luciferase and rhodanese, which we found to be differently processed by the Hsp70 chaperone machinery and whose conformational properties were investigated by biophysical approaches. In spite of their monomeric nature, they displayed enhanced thioflavin T fluorescence, non-native β-sheets, and tertiary structures with surface-accessible hydrophobic patches, but differed in their conformational stability and aggregation propensity. Interestingly, minor structural differences between the two misfolded species could account for their markedly different behavior in chaperone-mediated unfolding/refolding assays. Indeed, only a single DnaK molecule was sufficient to unfold by direct clamping a misfolded luciferase monomer, while, by contrast, several DnaK molecules were necessary to unfold the more resistant misfolded rhodanese monomer by a combination of direct clamping and cooperative entropic pulling.

Human immunoglobulins and Fc fragments promote microtubule assembly via tau proteins and induce conformational changes of neuronal microtubules in vitro.

The influence of human immunoglobulins (Ig) in neuronal cytoskeleton stability was studied in vitro. Here we show that human Ig and Fc fragments stimulate animal and human microtubule assembly by binding to microtubules via tau isoforms. In presence of Ig, microtubules show increased aggregation, twisting and rigidity. Non-immune Ig and Fc fragments promote microtubule assembly in temperature-dependent manner and stabilize microtubules at a molecular ratio of 1 Ig per 4 tubulin dimers. These in vitro data provide an experimental support for an immuno-mediated modulation of the cytoskeleton. In conjunction with previous neuropathological data, they suggest that Ig could participate in early stages of neurodegeneration by affecting the microtubule stability in vivo.

Study of biomacromolecule conformational changes by Scanning Force Microscopy

L?objectif de ce travail de thèse est l?étude des changements conformationels des biomacromolecules à l?échelle d?une molécule unique. Pour cela on a utilisé la Microscopie à Force Atomique (AFM) appliqué à l?étude des protéines et des acides nucléiques déposés sur une surface. Dans ce type de microscopie, une pointe très fine attachée à l?extrémité d?un levier est balayée au dessus d?une surface. L?interaction de la pointe avec la surface de l?échantillon induit la déflection du levier et ce phénomène permet de reconstruire la topographie de l?échantillon. Très importante dans cette technique est la possibilité de travailler en liquide. Cela permet de étudier les biomolécules en conditions quasi-physiologiques sans qu?elles perdent leur activité. On a étudié GroEL, la chaperonin de E.coli, qui est un homo oligomère avec une structure à double anneau qui joue un rôle très important dans le repliement des protéines dénaturées et celles qui viennent d?être synthétisées. En particulier on a focalisé notre attention sur la stabilité mécanique et sur les changements conformationels qui ont lieu pendant l?activité de GroEL. Une analyse détaillée des changements dans la stabilité mécanique et des effets produits par la liaison et l?hydrolyse de l?ATP est présentée dans ce travail. On a montré que le point le plus faible dans la structure de GroEL est l?interface entre les deux anneaux et que l?étape critique dans l?affaiblissement de la structure est l?hydrolyse de l?ATP. En ce qui concerne le changement conformationel, le passage d?une surface hydrophobe à hydrophile, induit par l?hydrolyse de l?ATP, a été montré. Ensuite on a étudié le changement dans la conformation et dans la topologie de l?ADN résultant de l?interaction avec des molécules spécifiques et en réponse à l?exposition des cellules de E.coli à des conditions de stress. Le niveau de surenroulement est un paramètre très sensible, de façon variée, à tous ces facteurs. Les cellules qui ont crus à de températures plus élevées que leur température optimale ont la tendance à diminuer le nombre de surenroulements négatif pour augmenter la stabilité thermique de leur plasmides. L?interaction avec des agents intercalant induit une transition d?un surenroulement négatif à un surenroulement positif d?une façon dépendante de la température. Finalement, l?effet de l?interaction de l?ADN avec des surfaces différentes a été étudié et une application pratique sur les noeuds d?ADN est présentée.<br/><br/>The aim of the present thesis work is to study the conformational changes of biomacromolecules at the single molecule level. To that end, Atomic Force Microcopy (AFM) imaging was performed on proteins and nucleic acids adsorbed onto a surface. In this microcopy technique a very sharp tip attached at the end of a soft cantilever is scanned over a surface, the interaction of the tip with the sample?s surface will induce the deflection of the cantilever and thus it will make possible to reconstruct the topography. A very important feature of AFM is the possibility to operate in liquid, it means with the sample immersed in a buffer solution. This allows one to study biomolecules in quasi-physiological conditions without loosing their activity. We have studied GroEL, the chaperonin of E.coli, which is a double-ring homooligomer which pays a very important role in the refolding of unfolded and newly synthetized polypeptides. In particular we focus our attention on its mechanical stability and on the conformational change that it undergoes during its activity cycle. A detailed analysis of the change in mechanical stability and how it is affected by the binding and hydrolysis of nucleotides is presented. It has been shown that the weak point of the chaperonin complex is the interface between the two rings and that the critical step to weaken the structure is the hydrolysis of ATP. Concerning the conformational change we have directly measured, with a nanometer scale resolution, the switching from a hydrophobic surface to a hydrophilic one taking place inside its cavity induced by the ATP hydrolysis. We have further studied the change in the DNA conformation and topology as a consequence of the interaction with specific DNA-binding molecules and the exposition of the E.coli cells to stress conditions. The level of supercoiling has been shown to be a very sensitive parameter, even if at different extents, to all these factors. Cells grown at temperatures higher than their optimum one tend to decrease the number of the negative superhelical turns in their plasmids in order to increase their thermal stability. The interaction with intercalating molecules induced a transition from positive to negative supercoiling in a temperature dependent way. The effect of the interaction of the DNA with different surfaces has been investigated and a practical application to DNA complex knots is reported.<br/><br/>Observer les objets biologiques en le touchant Schématiquement le Microscope a Force Atomique (AFM) consiste en une pointe très fine fixée a l?extrémité d?un levier Lors de l?imagerie, la pointe de l?AFM gratte la surface de l?échantillon, la topographie de celui-ci induit des déflections du levier qui sont enregistrées au moyen d?un rayon laser réfléchi par le levier. Ces donnés sont ensuit utilisés par un ordinateur pour reconstituer en 3D la surface de l?échantillon. La résolution de l?instrument est fonction entre autre de la dureté, de la rugosité de l?échantillon et de la forme de la pointe. Selon l?échantillon et la pointe utilisée la résolution de l?AFM peut aller de 0.1 A (sur des cristaux) a quelque dizaine de nanomètres (sur des cellules). Cet instrument est particulierment intéressant en biologie en raison de sa capacité à imager des échantillons immergés dans un liquide, c?est à dire dans des conditions quasiphysiologiques. Dans le cadre de ce travail nous avons étudié les changements conformationels de molécules biologiques soumises à des stimulations externes. Nous avons essentielment concentré notre attention sur des complexes protéiques nommé Chaperons Moléculaires et sur des molécules d?ADN circulaire (plasmides). Les Chaperons sont impliqués entre autre dans la résistance des organismes vivants aux stress thermiques et osmotiques. Leur activité consiste essentielment à aider les autres protéines à être bien pliés dans leur conformation finale et, en conséquence, à eviter que ils soient dénaturées et que ils puissent s?agréger. L?ADN, quant à lui est la molécule qui conserve, dans sa séquence, l?information génétique de tous les organismes vivants. Ce travail a spécifiquement concerné l?étude des changements conformationels des chaperonins suit a leur activation par l?ATP. Ces travaux ont montrés a l?échelle de molécule unique la capacité de ces protéines de changer leur surface de hydrophobique a hydrophilique. Nous avons également utilisé l?AFM pour étudier le changement du nombre des surenroulements des molécules d?ADN circulaire lors d?une exposition à un changement de température et de force ionique. Ces travaux ont permis de montrer comment la cellule regle le nombre de surenroulements dans ces molécules pour répondre et contrôler l?expression génétique même dans de conditions extrêmes. Pour les deux molécules en général, c?était très important d?avoir la possibilité de observer leur transitions d?une conformation a l?autre directement a l?échelle d?une seul molécule et, surtout, avec une résolution largement au dessous des la longueur d?onde de la lumière visible que représente le limite pour l?imagerie optique.

Regulation of Nedd4-2 self-ubiquitination and stability by a PY motif located within its HECT-domain.

Ubiquitin ligases play a pivotal role in substrate recognition and ubiquitin transfer, yet little is known about the regulation of their catalytic activity. Nedd4 (neural-precursor-cell-expressed, developmentally down-regulated 4)-2 is an E3 ubiquitin ligase composed of a C2 domain, four WW domains (protein-protein interaction domains containing two conserved tryptophan residues) that bind PY motifs (L/PPXY) and a ubiquitin ligase HECT (homologous with E6-associated protein C-terminus) domain. In the present paper we show that the WW domains of Nedd4-2 bind (weakly) to a PY motif (LPXY) located within its own HECT domain and inhibit auto-ubiquitination. Pulse-chase experiments demonstrated that mutation of the HECT PY-motif decreases the stability of Nedd4-2, suggesting that it is involved in stabilization of this E3 ligase. Interestingly, the HECT PY-motif mutation does not affect ubiquitination or down-regulation of a known Nedd4-2 substrate, ENaC (epithelial sodium channel). ENaC ubiquitination, in turn, appears to promote Nedd4-2 self-ubiquitination. These results support a model in which the inter- or intra-molecular WW-domain-HECT PY-motif interaction stabilizes Nedd4-2 by preventing self-ubiquitination. Substrate binding disrupts this interaction, allowing self-ubiquitination of Nedd4-2 and subsequent degradation, resulting in down-regulation of Nedd4-2 once it has ubiquitinated its target. These findings also point to a novel mechanism employed by a ubiquitin ligase to regulate itself differentially compared with substrate ubiquitination and stability.

Accurate performance of a rat model of schizophrenia in the water maze depends on visual cue availability and stability: a distortion in cognitive mapping abilities?

Rats were treated postnatally (PND 5-16) with BSO (l-buthionine-(S,R)-sulfoximine) in an animal model of schizophrenia based on transient glutathione deficit. The BSO treated rats were impaired in patrolling a maze or a homing table when adult, yet demonstrated preserved escape learning, place discrimination and reversal in a water maze task [37]. In the present work, BSO rats' performance in the water maze was assessed in conditions controlling for the available visual cues. First, in a completely curtained environment with two salient controlled cues, BSO rats showed little accuracy compared to control rats. Secondly, pre-trained BSO rats were impaired in reaching the familiar spatial position when curtains partially occluded different portions of the room environment in successive sessions. The apparently preserved place learning in a classical water maze task thus appears to require the stability and the richness of visual landmarks from the surrounding environment. In other words, the accuracy of BSO rats in place and reversal learning is impaired in a minimal cue condition or when the visual panorama changes between trials. However, if the panorama remains rich and stable between trials, BSO rats are equally efficient in reaching a familiar position or in learning a new one. This suggests that the BSO accurate performance in the water maze does not satisfy all the criteria for a cognitive map based navigation on the integration of polymodal cues. It supports the general hypothesis of a binding deficit in BSO rats.

Three-dimensional slope stability analysis of South Peak, Crowsnest Pass, Alberta, Canada

South Peak is a 7-Mm3 potentially unstable rock mass located adjacent to the 1903 Frank Slide on Turtle Mountain, Alberta. This paper presents three-dimensional numerical rock slope stability models and compares them with a previous conceptual slope instability model based on discontinuity surfaces identified using an airborne LiDAR digital elevation model (DEM). Rock mass conditions at South Peak are described using the Geological Strength Index and point load tests, whilst the mean discontinuity set orientations and characteristics are based on approximately 500 field measurements. A kinematic analysis was first conducted to evaluate probable simple discontinuity-controlled failure modes. The potential for wedge failure was further assessed by considering the orientation of wedge intersections over the airborne LiDAR DEM and through a limit equilibrium combination analysis. Block theory was used to evaluate the finiteness and removability of blocks in the rock mass. Finally, the complex interaction between discontinuity sets and the topography within South Peak was investigated through three-dimensional distinct element models using the code 3DEC. The influence of individual discontinuity sets, scale effects, friction angle and the persistence along the discontinuity surfaces on the slope stability conditions were all investigated using this code.

Gains from switching and evolutionary stability in multi-player matrix games.

In this paper we unify, simplify, and extend previous work on the evolutionary dynamics of symmetric N-player matrix games with two pure strategies. In such games, gains from switching strategies depend, in general, on how many other individuals in the group play a given strategy. As a consequence, the gain function determining the gradient of selection can be a polynomial of degree N-1. In order to deal with the intricacy of the resulting evolutionary dynamics, we make use of the theory of polynomials in Bernstein form. This theory implies a tight link between the sign pattern of the gains from switching on the one hand and the number and stability of the rest points of the replicator dynamics on the other hand. While this relationship is a general one, it is most informative if gains from switching have at most two sign changes, as is the case for most multi-player matrix games considered in the literature. We demonstrate that previous results for public goods games are easily recovered and extended using this observation. Further examples illustrate how focusing on the sign pattern of the gains from switching obviates the need for a more involved analysis.

Ligand binding transmits conformational changes across the membrane-spanning region to the intracellular side of the 5-HT3 serotonin receptor.

Conformational changes of channel activation: Five enhanced green fluorescent protein (EGFP) molecules (green cylinders) were integrated into the intracellular part of the homopentameric ionotropic 5-HT3 receptor. This allowed the detection of extracellular binding of fluorescent ligands (?) to EGFP by FRET, and also enabled the quantification of agonist-induced conformational changes in the intracellular region of the receptor by homo-FRET between EGFPs. The approach opens novel ways for probing receptor activation and functional screening of therapeutic compounds.

Stability analysis of the 2007 Chehalis lake landslide based on long-range terrestrial photogrammetry and airborne LIDAR data

On December 4th 2007, a 3-Mm3 landslide occurred along the northwestern shore of Chehalis Lake. The initiation zone is located at the intersection of the main valley slope and the northern sidewall of a prominent gully. The slope failure caused a displacement wave that ran up to 38 m on the opposite shore of the lake. The landslide is temporally associated with a rain-on-snow meteorological event which is thought to have triggered it. This paper describes the Chehalis Lake landslide and presents a comparison of discontinuity orientation datasets obtained using three techniques: field measurements, terrestrial photogrammetric 3D models and an airborne LiDAR digital elevation model to describe the orientation and characteristics of the five discontinuity sets present. The discontinuity orientation data are used to perform kinematic, surface wedge limit equilibrium and three-dimensional distinct element analyses. The kinematic and surface wedge analyses suggest that the location of the slope failure (intersection of the valley slope and a gully wall) has facilitated the development of the unstable rock mass which initiated as a planar sliding failure. Results from the three-dimensional distinct element analyses suggest that the presence, orientation and high persistence of a discontinuity set dipping obliquely to the slope were critical to the development of the landslide and led to a failure mechanism dominated by planar sliding. The three-dimensional distinct element modelling also suggests that the presence of a steeply dipping discontinuity set striking perpendicular to the slope and associated with a fault exerted a significant control on the volume and extent of the failed rock mass but not on the overall stability of the slope.

Dimensionality of drinking consequences - cross-cultural comparability and stability over time

Despite the long tradition for asking about the negative social and health consequences of alcohol consumption in surveys, little is known about the dimensionality of these consequences. Analysing cross-sectional and longitudinal data from the Nordic Taxation Study collected for Sweden, Finland, and Denmark in two waves in 2003 and 2004 by means of an explorative principal component analysis for categorical data (CATPCA), it is tested whether consequences have a single underlying dimension across cultures. It further tests the reliability, replicability, concurrent and predictive validity of the consequence scales. A one-dimensional solution was commonly preferable. Whereas the two-dimensional solution was unable to distinguish clearly between different concepts of consequences, the one-dimensional solution resulted in interpretable, generally very stable scales within countries across different samples and time.

Stability and change in party preferences.

This article analyses stability and volatility of party preferences using data from the Swiss Household-Panel (SHP), which, for the first time, allow studying transitions and stability of voters over several years in Switzerland. Analyses cover the years 1999- 2007 and systematically distinguish changes between party blocks and changes within party blocks. The first part looks at different patterns of change, which show relatively high volatility. The second part tests several theories on causes of such changes applying a multinomial random-effects model. Results show that party preferences stabilise with their duration and with age and that the electoral cycle, political sophistication, socio-structural predispositions, the household-context as well as party size and the number of parties each explain part of electoral volatility. Different results for withinand between party-block changes underlie the importance of that differentiation.