Cyclic olefin homopolymer-based microfluidics for protein crystallization and in situ X-ray diffraction

A cyclic olefin homopolymer-based microfluidics system has been established for protein crystallization and in situ X-ray diffraction

Mutantelec: AnIn Silicomutation simulation platform for comparative electrostatic potential profiling of proteins

The electrostatic potential plays a key role in many biological processes like determining the affinity of a ligand to a given protein target, and they are responsible for the catalytic activity of many enzymes. Understanding the effect that amino acid mutations will have on the electrostatic potential of a protein, will allow a thorough understanding of which residues are the most important in a protein. MutantElec, is a friendly web application for in silico generation of site-directed mutagenesis of proteins and the comparison of electrostatic potential between the wild type protein and the mutant(s), based on the three-dimensional structure of the protein. The effect of the mutation is evaluated using different approach to the traditional surface map. MutantElec provides a graphical display of the results that allows the visualization of changes occurring at close distance from the mutation and thus uncovers the local and global impact of a specific chang

Motif co-regulation and co-operativity are common mechanisms in transcriptional, post-transcriptional and post-translational regulation

A substantial portion of the regulatory interactions in the higher eukaryotic cell are mediated by simple sequence motifs in the regulatory segments of genes and (pre-)mRNAs, and in the intrinsically disordered regions of proteins. Although these regulatory modules are physicochemically distinct, they share an evolutionary plasticity that has facilitated a rapid growth of their use and resulted in their ubiquity in complex organisms. The ease of motif acquisition simplifies access to basal housekeeping functions, facilitates the co-regulation of multiple biomolecules allowing them to respond in a coordinated manner to changes in the cell state, and supports the integration of multiple signals for combinatorial decision-making. Consequently, motifs are indispensable for temporal, spatial, conditional and basal regulation at the transcriptional, post-transcriptional and post-translational level. In this review, we highlight that many of the key regulatory pathways of the cell are recruited by motifs and that the ease of motif acquisition has resulted in large networks of co-regulated biomolecules. We discuss how co-operativity allows simple static motifs to perform the conditional regulation that underlies decision-making in higher eukaryotic biological systems. We observe that each gene and its products have a unique set of DNA, RNA or protein motifs that encode a regulatory program to define the logical circuitry that guides the life cycle of these biomolecules, from transcription to degradation. Finally, we contrast the regulatory properties of protein motifs and the regulatory elements of DNA and (pre-)mRNAs, advocating that co-regulation, co-operativity, and motif-driven regulatory programs are common mechanisms that emerge from the use of simple, evolutionarily plastic regulatory modules

A conformational switch regulates p53 DNA binding

The p53 tumour suppressor gene encodes for a transcription factor that encompasses a sequence-specific DNA-binding and an oligomerization domains. This gene is the most frequently mutated gene in human cancers. It remains unclear how p53 recognizes its DNA target sites on the genome. We describe here the structures of p53 in absence and in complex with DNA. Structure and kinetic analyses of p53-DNA complexes reveal that p53 recognizes its specific DNA target sites via an induced fit mechanism. The affinities of p53 for specific and non-specific DNA are similar, but the off-rates are much different. The loop L1 of the DNA-binding domain of p53 undergoes a conformational switch. Overall, these results reveal the importance of the conformational switch of loop L1 in the DNA binding properties of p53. We also describe here the first structure of p53 in complex with the natural CDKN1A (p21) p53-response element

Édition critique et étude doctrinale des <i>Quaestiones super Sophisticos elenchos</i> de Radulphus Brito

Cette thèse de doctorat porte sur un commentaire médiéval des Réfutations sophistiques d’Aristote, un fameux traité dans lequel il expose les différents ressorts d’une argumentation fallacieuse. Ce commentaire, les Quaestiones super Sophisticos elenchos, est l’œuvre du dernier des plus grands maîtres ès Arts parisiens du XIIIe siècle, Radulphus Brito. La thèse est constituée de deux parties principales, une édition critique complète des QSE (sur la base de 4 manuscrits latins) et une étude doctrinale. Dans l’étude, j’ai cherché à reconstruire la définition du syllogisme sophistique selon Brito, en l’abordant selon quatre perspectives, à savoir ontologique, formelle, sémantique et « pragmatique », une perspective qui consiste à prendre en compte le contexte d’énonciation, les intentions et les interprétations des locuteurs. Il ressort de cette étude que Brito est un témoin majeur d’un tournant davantage orienté vers des considérations d’ordre pragmatique en logique à la fin du XIIIe siècle

Crystal Structure of a Multidomain Human p53 Tetramer Bound to the Natural <i>CDKN1A</i> (<i>p21</i>) p53-Response Element

The p53 tumor suppressor protein is a sequence-specific DNA-binding transcription factor. Structures of p53 bound to DNA have been described, but, so far, no structure has been determined of p53 bound to a natural p53-response element. We describe here the structure of a human p53 homotetramer encompassing both the DNA-binding and homo-oligomerization domains in complex with the natural p53-response element present upstream of the promoter of the CDKN1A (p21) gene. Similar to our previously described structures of human p53 tetramers bound to an artificial consensus DNA site, p53 DNA binding proceeds via an induced fit mechanism with loops L1 of two subunits adopting recessed conformations. Interestingly, the conformational change involving loop L1 is even more extreme than the one previously observed with the artificial consensus DNA site. In fact, the previously determined loop L1 conformation seems to be a transition intermediate between the non–DNA-bound and CDKN1A-bound states. Thus, the new structure further supports our model that recognition of specific DNA by p53 is associated with conformational changes within the DNA-binding domain of p53. Mol Cancer Res; 9(11); 1493–9. ©2011 AACR.</p

Reversal of the DNA-Binding-Induced Loop L1 Conformational Switch in an Engineered Human p53 Protein

The gene encoding the p53 tumor suppressor protein, a sequence-specific DNA binding transcription factor, is the most frequently mutated gene in human cancer. Crystal structures of homo-oligomerizing p53 polypeptides with specific DNA suggest that DNA binding is associated with a conformational switch. Specifically, in the absence of DNA, loop L1 of the p53 DNA binding domain adopts an extended conformation, whereas two p53 subunits switch to a recessed loop L1 conformation when bound to DNA as a tetramer. We previously designed a p53 protein, p53FG, with amino substitutions S121F and V122G targeting loop L1. These two substitutions enhanced the affinity of p53 for specific DNA yet, counterintuitively, decreased the residency time of p53 on DNA. Here, we confirmed these DNA binding properties of p53FG using a different method. We also determined by crystallography the structure of p53FG in its free state and bound to DNA as a tetramer. In the free state, loop L1 adopted a recessed conformation, whereas upon DNA binding, two subunits switched to the extended loop L1 conformation, resulting in a final structure that was very similar to that of wild-type p53 bound to DNA. Thus, altering the apo structure of p53 changed its DNA binding properties, even though the DNA-bound structure was not altered.</p

Expression, crystallization and preliminary X-ray diffraction analysis of the CMM2 region of the Arabidopsis thaliana Morpheus' molecule 1 protein

Of the known epigenetic control regulators found in plants, the Morpheus' molecule 1 (MOM1) protein is atypical in that the deletion of MOM1 does not affect the level of epigenetic marks controlling the transcriptional status of the genome. A short 197-amino-acid fragment of the MOM1 protein sequence can complement MOM1 deletion when coupled to a nuclear localization signal, suggesting that this region contains a functional domain that compensates for the loss of the full-length protein. Numerous constructs centred on the highly conserved MOM1 motif 2 (CMM2) present in these 197 residues have been generated and expressed in Escherichia coli. Following purification and crystallization screening, diamond-shaped single crystals were obtained that diffracted to approximately 3.2 A resolution. They belonged to the trigonal space group P3(1)21 (or P3(2)21), with unit-cell parameters a=85.64, c=292.74 A. Structure determination is ongoing

Reactivation of mutant p53: Constraints on mechanism highlighted by principal component analysis of the DNA binding domain

Most p53 mutations associated with cancer are located in its DNA binding domain (DBD). Many structures (X-ray and NMR) of this domain are available in the protein data bank (PDB) and a vast conformational heterogeneity characterizes the various free and complexed states. The major difference between the apo and the holo-complexed states appears to lie in the L1 loop. In particular, the conformations of this loop appear to depend intimately on the sequence of DNA to which it binds. This conclusion builds upon recent observations that implicate the tetramerization and the C-terminal domains (respectively TD and Cter) in DNA binding specificity. Detailed PCA analysis of the most recent collection of DBD structures from the PDB have been carried out. In contrast to recommendations that small molecules/drugs stabilize the flexible L1 loop to rescue mutant p53, our study highlights a need to retain the flexibility of the p53 DNA binding surface (DBS). It is the adaptability of this region that enables p53 to engage in the diverse interactions responsible for its functionalit