Mean first passage time analysis reveals rate-limiting steps, parallel pathways and dead ends in a simple model of protein folding

We have analyzed dynamics on the complex free energy landscape of protein folding in the FOLD-X model, by calculating for each state of the system the mean first passage time to the folded state. The resulting kinetic map of the folding process shows that it proceeds in jumps between well-defined, local free energy minima. Closer analysis of the different local minima allows us to reveal secondary, parallel pathways as well as dead ends.Comment: 7 page

Thermodynamic Prediction of Protein Neutrality

We present a simple theory that uses thermodynamic parameters to predict the probability that a protein retains the wildtype structure after one or more random amino acid substitutions. Our theory predicts that for large numbers of substitutions the probability that a protein retains its structure will decline exponentially with the number of substitutions, with the severity of this decline determined by properties of the structure. Our theory also predicts that a protein can gain extra robustness to the first few substitutions by increasing its thermodynamic stability. We validate our theory with simulations on lattice protein models and by showing that it quantitatively predicts previously published experimental measurements on subtilisin and our own measurements on variants of TEM1 beta-lactamase. Our work unifies observations about the clustering of functional proteins in sequence space, and provides a basis for interpreting the response of proteins to substitutions in protein engineering applications

Transition states in protein folding kinetics: Modeling Phi-values of small beta-sheet proteins

Small single-domain proteins often exhibit only a single free-energy barrier, or transition state, between the denatured and the native state. The folding kinetics of these proteins is usually explored via mutational analysis. A central question is which structural information on the transition state can be derived from the mutational data. In this article, we model and structurally interpret mutational Phi-values for two small beta-sheet proteins, the PIN and the FBP WW domain. The native structure of these WW domains comprises two beta-hairpins that form a three-stranded beta-sheet. In our model, we assume that the transition state consists of two conformations in which either one of the hairpins is formed. Such a transition state has been recently observed in Molecular Dynamics folding-unfolding simulations of a small designed three-stranded beta-sheet protein. We obtain good agreement with the experimental data (i) by splitting up the mutation-induced free-energy changes into terms for the two hairpins and for the small hydrophobic core of the proteins, and (ii) by fitting a single parameter, the relative degree to which hairpin 1 and 2 are formed in the transition state. The model helps to understand how mutations affect the folding kinetics of WW domains, and captures also negative Phi-values that have been difficult to interpret.Comment: 27 pages, 6 pages, 3 tables; to appear in Biophys.

Structural and functional analysis of SGT1–HSP90 core complex required for innate immunity in plants

SGT1 (Suppressor of G2 allele of skp1), a co-chaperone of HSP90 (Heat-shock protein 90), is required for innate immunity in plants and animals. Unveiling the cross talks between SGT1 and other co-chaperones such as p23, AHA1 (Activator of HSP90 ATPase 1) or RAR1 (Required for Mla12 resistance) is an important step towards understanding the HSP90 machinery. Nuclear magnetic resonance spectroscopy and mutational analyses of HSP90 revealed the nature of its binding with the CS domain of SGT1. Although CS is structurally similar to p23, these domains were found to non-competitively bind to various regions of HSP90; yet, unexpectedly, full-length SGT1 could displace p23 from HSP90. RAR1 partly shares the same binding site with HSP90 as the CS domain, whereas AHA1 does not. This analysis allowed us to build a structural model of the HSP90–SGT1 complex and to obtain a compensatory mutant pair between both partners that is able to restore virus resistance in vivo through Rx (Resistance to potato virus X) immune sensor stabilization

Trajectoires résidentielles, construction des espaces de vie et ancrage dans le périurbain. Enquête au nord de l’agglomération parisienne

International audienceThis article focuses on the links between residential trajectories, building living spaces, and territorial anchoring in suburban areas. It follows recent studies that qualify certain homogenizing representations of suburban residents. It uses the results of a survey conducted in the suburban area north of the greater Paris region. This survey consisted of around one hundred interviews with households of varied social positions, concerning their residential and biographical history, their practices, and representations of their living space. Beyond the image of the dual income family leaving the dense city to buy a house in an improved living environment, the article reflects the diversity of the types of inhabitants’ residential trajectories and of the logic explaining their residential choice. It not only stresses that the living spaces of suburban inhabitants are characterized by proximity to, and the use of, local resources, but, in addition, points out that previous places of residence are also resource sites for individuals, indicating a “multi-polarization” of practices and a “multi-anchoring” of individuals.Cet article aborde les liens entre trajectoires résidentielles, construction des espaces de vie et ancrage territorial dans les espaces périurbains. Il s’inscrit dans la lignée de travaux récents qui nuancent certaines représentations tendant à homogénéiser les habitants du périurbain. Il exploite les résultats d’une enquête menée dans le périurbain au nord de l’agglomération parisienne, qui a consisté en une centaine d’entretiens avec des ménages aux positions sociales variées, portant sur leurs parcours résidentiel et biographique, leurs pratiques et représentations de leur espace de vie. Au-delà de la figure du couple bi-actif qui quitte la ville dense pour acheter une maison dans un cadre de vie valorisé, l’article rend compte de la diversité des types de trajectoires résidentielles de ces habitants et des logiques expliquant leur choix résidentiel. Il souligne également que les espaces de vie des habitants du périurbain sont caractérisés par la proximité et le recours aux ressources locales, mais que les lieux de résidences antérieures constituent des lieux de ressources pour les individus, ce qui induit une « multipolarisation » des pratiques et un « multi-ancrage »

From Urban Morphological Zones (UMZ) to harmonised «urban objects » in Europe

International audienceUntil now, the building of comparable databases for European cities has been made through two different approaches: - a bottom up process, from national to European level, relying on the collect of national delineations by European institutions (NUREC 1994, ESPON 1.4.3. 2007, Urban Audit 2009) - a top down process, which consists in applying the same criteria to the whole European space (e.g. Urban Morphological Zones, produced by the European Agency of Environment, 2000). In the latter case it is essential to submit the resulting objects to a validation process worked out by national experts or based on national databases. Our paper is a contribution to the validation of UMZ database, in order to make these morphological zones more operational for urban studies. We develop here a first expertise by comparing UMZ to French and Danish morphological agglomerations. We first introduce the methodological frame of this comparison, which has been thought to be transposed to other countries. This protocol has to consider semantic and geometric differences between databases. It leads to a few fittings in order to enable the integration of both UMZ and national urban databases. Then we develop the main results of the comparison made in France and Denmark, from both quantitative and qualitative points of view. The measure of key indicators underlines the convergence of UMZ and national databases (an average difference of +/- 5% for urban populations). However, in France some large cities are sometimes much more spread out in national databases than they are from UMZ source. These main types of differences in France can be due to specific types of settlement patterns (coastal or industrial conurbations, large city margins). It raises the issue of applying similar criteria in different territorial contexts.By improving our knowledge about UMZ and their use for urban studies, this expertise aims at constructing comparable databases and it follows practical goals. But it also tends to enlighten the specificities of some settlement patterns throughout Europe, in a more exploratory way

Experimental library screening demonstrates the successful application of computational protein design to large structural ensembles

The stability, activity, and solubility of a protein sequence are determined by a delicate balance of molecular interactions in a variety of conformational states. Even so, most computational protein design methods model sequences in the context of a single native conformation. Simulations that model the native state as an ensemble have been mostly neglected due to the lack of sufficiently powerful optimization algorithms for multistate design. Here, we have applied our multistate design algorithm to study the potential utility of various forms of input structural data for design. To facilitate a more thorough analysis, we developed new methods for the design and high-throughput stability determination of combinatorial mutation libraries based on protein design calculations. The application of these methods to the core design of a small model system produced many variants with improved thermodynamic stability and showed that multistate design methods can be readily applied to large structural ensembles. We found that exhaustive screening of our designed libraries helped to clarify several sources of simulation error that would have otherwise been difficult to ascertain. Interestingly, the lack of correlation between our simulated and experimentally measured stability values shows clearly that a design procedure need not reproduce experimental data exactly to achieve success. This surprising result suggests potentially fruitful directions for the improvement of computational protein design technology

HotPoint: hot spot prediction server for protein interfaces

The energy distribution along the protein–protein interface is not homogenous; certain residues contribute more to the binding free energy, called ‘hot spots’. Here, we present a web server, HotPoint, which predicts hot spots in protein interfaces using an empirical model. The empirical model incorporates a few simple rules consisting of occlusion from solvent and total knowledge-based pair potentials of residues. The prediction model is computationally efficient and achieves high accuracy of 70%. The input to the HotPoint server is a protein complex and two chain identifiers that form an interface. The server provides the hot spot prediction results, a table of residue properties and an interactive 3D visualization of the complex with hot spots highlighted. Results are also downloadable as text files. This web server can be used for analysis of any protein–protein interface which can be utilized by researchers working on binding sites characterization and rational design of small molecules for protein interactions. HotPoint is accessible at http://prism.ccbb.ku.edu.tr/hotpoint

Dynamics of Ku and bacterial non-homologous end-joining characterized using single DNA molecule analysis

We use single-molecule techniques to characterize the dynamics of prokaryotic DNA repair by non-homologous end-joining (NHEJ), a system comprised only of the dimeric Ku and Ligase D (LigD). The Ku homodimer alone forms a ∼2 s synapsis between blunt DNA ends that is increased to ∼18 s upon addition of LigD, in a manner dependent on the C-terminal arms of Ku. The synapsis lifetime increases drastically for 4 nt complementary DNA overhangs, independently of the C-terminal arms of Ku. These observations are in contrast to human Ku, which is unable to bridge either of the two DNA substrates. We also demonstrate that bacterial Ku binds the DNA ends in a cooperative manner for synapsis initiation and remains stably bound at DNA junctions for several hours after ligation is completed, indicating that a system for removal of the proteins is active in vivo. Together these experiments shed light on the dynamics of bacterial NHEJ in DNA end recognition and processing. We speculate on the evolutionary similarities between bacterial and eukaryotic NHEJ and discuss how an increased understanding of bacterial NHEJ can open the door for future antibiotic therapies targeting this mechanism