Stochastic protein folding simulation in the three-dimensional HP-model

We present results from three-dimensional protein folding simulations in the HP-model on ten benchmark problems. The simulations are executed by a simulated annealing-based algorithm with a time-dependent cooling schedule. The neighbourhood relation is determined by the pull-move set. The results provide experimental evidence that the maximum depth D of local minima of the underlying energy landscape can be upper bounded by D < n(2/3). The local search procedure employs the stopping criterion (In/delta)(D/gamma) where m is an estimation of the average number of neighbouring conformations, gamma relates to the mean of non-zero differences of the objective function for neighbouring conformations, and 1-delta is the confidence that a minimum conformation has been found. The bound complies with the results obtained for the ten benchmark problems. (c) 2008 Elsevier Ltd. All rights reserved.

Population-based local search for protein folding simulation in the MJ energy model and cubic lattices

We present experimental results on benchmark problems in 3D cubic lattice structures with the Miyazawa-Jernigan energy function for two local search procedures that utilise the pull-move set: (i) population-based local search (PLS) that traverses the energy landscape with greedy steps towards (potential) local minima followed by upward steps up to a certain level of the objective function; (ii) simulated annealing with a logarithmic cooling schedule (LSA). The parameter settings for PLS are derived from short LSA-runs executed in pre-processing and the procedure utilises tabu lists generated for each member of the population. In terms of the total number of energy function evaluations both methods perform equally well, however. PLS has the potential of being parallelised with an expected speed-up in the region of the population size. Furthermore, both methods require a significant smaller number of function evaluations when compared to Monte Carlo simulations with kink-jump moves. (C) 2009 Elsevier Ltd. All rights reserved.

Approximating the Set of Local Minima in Partial RNA Folding Landscapes

Motivation: We study a stochastic method for approximating the set of local minima in partial RNA folding landscapes associated with a bounded-distance neighbourhood of folding conformations. The conformations are limited to RNA secondary structures without pseudoknots. The method aims at exploring partial energy landscapes pL induced by folding simulations and their underlying neighbourhood relations. It combines an approximation of the number of local optima devised by Garnier and Kallel (2002) with a run-time estimation for identifying sets of local optima established by Reeves and Eremeev (2004).

Results: The method is tested on nine sequences of length between 50 nt and 400 nt, which allows us to compare the results with data generated by RNAsubopt and subsequent barrier tree calculations. On the nine sequences, the method captures on average 92% of local minima with settings designed for a target of 95%. The run-time of the heuristic can be estimated by O(n2D?ln?), where n is the sequence length, ? is the number of local minima in the partial landscape pL under consideration and D is the maximum number of steepest descent steps in attraction basins associated with pL.

Facilitating folding of outer membrane proteins, roles of the periplasmic chaperone Skp and the outer membrane lipoprotein BamD

This thesis describes several important advancements in the understanding of the assembly of outer membrane proteins of Gram-negative bacteria like Escherichia coli. A first study was performed to identify binding regions in the trimeric chaperone Skp for outer membrane proteins. Skp is known to facilitate the passage of unfolded outer membrane proteins (OMPs) through the periplasm to the outer membrane (OM). A gene construct named “synthetic chaperone protein (scp)” gene was used to express a fusion protein (Scp) into the cytoplasm of E. coli. The scp gene was used as a template to design mutants of Scp suitable for structural and functional studies using site-directed spectroscopy. Fluorescence resonance energy transfer (FRET) was used to identify distances in Skp-OmpA complexes that separate regions in Scp and in outer membrane protein A (OmpA) from E. coli. For this study, single cysteine (Cys) mutants and single Cys - single tryptophan (Trp) double mutants of Scp were prepared. For FRET experiments, the cysteines were labeled with the tryptophan fluorescence energy acceptor IAEDANS. Single Trp mutants of OmpA were used as fluorescence energy donors. In the second part of this thesis, the function of BamD and the structure of BamD-Scp complexes were examined. BamD is an essential component of the β-barrel assembly machinery (BAM) complex of the OM of Gram-negative bacteria. Fluorescence spectroscopy was used to probe the interactions of BamD with lipid membranes and to investigate the interactions of BamD with possible partner proteins from the periplasm and from the OM. A range of single cysteine (Cys) and single tryptophan (Trp) mutants of BamD were prepared. A very important conclusion from the extensive FRET study is that the essential lipoprotein BamD interacts and binds to the periplasmic chaperone Skp. BamD contains tetratrico peptide repeat (TPR) motifs that are suggested to serve as docking sites for periplasmic chaperones such as Skp.

The fisherman and his wife.

Érase una vez un pescador y su esposa que vivían cerca del mar, pero ella no era feliz en su pequeña cabaña. Un día, su marido le cuenta que ha pescado un pez encantado que habla y puede hacer realidad un deseo. La mujer le dice que pida una casita más grander.

Structure and biogenesis of the capsular F1 antigen from Yersinia pestis: Preserved folding energy drives fiber formation

Most gram-negative pathogens express fibrous adhesive virulence organelles that mediate targeting to the sites of infection. The F1 capsular antigen from the plague pathogen Yersinia pestis consists of linear fibers of a single subunit (Caf1) and serves as a prototype for nonpilus organelles assembled via the chaperone/usher pathway. Genetic data together with high-resolution X-ray structures corresponding to snapshots of the assembly process reveal the structural basis of fiber formation. Comparison of chaperone bound Caf1 subunit with the subunit in the fiber reveals a novel type of conformational change involving the entire hydrophobic core of the protein. The observed conformational change suggests that the chaperone traps a high-energy folding intermediate of Caf1. A model is proposed in which release of the subunit allows folding to be completed, driving fiber formation.

Limitation of double folding potentials to simulate the polarization in reactions involving halo nuclei

In this work, we investigate the limitation of the use of strength coefficients on double folding potentials to study the presence of the threshold anomaly in the elastic scattering of halo nuclei at near barrier energies. For this purpose, elastic angular distributions and reaction cross sections for the He-6 on Bi-209 are studied. (c) 2008 Elsevier B.V. All rights reserved.

A short proregion of trialysin, a pore-forming protein of Triatoma infestans salivary glands, controls activity by folding the N-terminal lytic motif

Triatoma infestans (Hemiptera: Reduviidae) is a hematophagous insect that transmits the protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas` disease. Its saliva contains trialysin, a protein that forms pores in membranes. Peptides based on the N-terminus of trialysin lyse cells and fold into alpha-helical amphipathic segments resembling antimicrobial peptides. Using a specific antiserum against trialysin, we show here that trialysin is synthesized as a precursor that is less active than the protein released after saliva secretion. A synthetic peptide flanked by a fluorophore and a quencher including the acidic proregion and the lytic N-terminus of the protein is also less active against cells and liposomes, increasing activity upon proteolysis. Activation changes the peptide conformation as observed by fluorescence increase and CD spectroscopy. This mechanism of activation could provide a way to impair the toxic effects of trialysin inside the salivary glands, thus restricting damaging lytic activity to the bite site.

The Xanthomonas citri effector protein PthA interacts with citrus proteins involved in nuclear transport, protein folding and ubiquitination associated with DNA repair

P>Xanthomonas axonopodis pv. citri utilizes the type III effector protein PthA to modulate host transcription to promote citrus canker. PthA proteins belong to the AvrBs3/PthA family and carry a domain comprising tandem repeats of 34 amino acids that mediates protein-protein and protein-DNA interactions. We show here that variants of PthAs from a single bacterial strain localize to the nucleus of plant cells and form homo- and heterodimers through the association of their repeat regions. We hypothesize that the PthA variants might also interact with distinct host targets. Here, in addition to the interaction with alpha-importin, known to mediate the nuclear import of AvrBs3, we describe new interactions of PthAs with citrus proteins involved in protein folding and K63-linked ubiquitination. PthAs 2 and 3 preferentially interact with a citrus cyclophilin (Cyp) and with TDX, a tetratricopeptide domain-containing thioredoxin. In addition, PthAs 2 and 3, but not 1 and 4, interact with the ubiquitin-conjugating enzyme complex formed by Ubc13 and ubiquitin-conjugating enzyme variant (Uev), required for K63-linked ubiquitination and DNA repair. We show that Cyp, TDX and Uev interact with each other, and that Cyp and Uev localize to the nucleus of plant cells. Furthermore, the citrus Ubc13 and Uev proteins complement the DNA repair phenotype of the yeast Delta ubc13 and Delta mms2/uev1a mutants, strongly indicating that they are also involved in K63-linked ubiquitination and DNA repair. Notably, PthA 2 affects the growth of yeast cells in the presence of a DNA damage agent, suggesting that it inhibits K63-linked ubiquitination required for DNA repair.