Junctions between i-motif tetramers in supramolecular structures

The symmetry of i-motif tetramers gives to cytidine-rich oligonucleotides the capacity to associate into supramolecular structures (sms). In order to determine how the tetramers are linked together in such structures, we have measured by gel filtration chromatography and NMR the formation and dissociation kinetics of sms built by oligonucleotides containing two short C stretches separated by a non-cytidine-base. We show that a stretch of only two cytidines either at the 3′- or 5′-end is long enough to link the tetramers into sms. The analysis of the properties of sms formed by oligonucleotides differing by the length of the oligo-C stretches, the sequence orientation and the nature of the non-C base provides a model of the junction connecting the tetramers in sms

Structural study of the membrane protein MscL using cell-free expression and solid-state

a b s t r a c t High-resolution structures of membrane proteins have so far been obtained mostly by X-ray crystallography, on samples where the protein is surrounded by detergent. Recent developments of solid-state NMR have opened the way to a new approach for the study of integral membrane proteins inside a membrane. At the same time, the extension of cell-free expression to the production of membrane proteins allows for the production of proteins tailor made for NMR. We present here an in situ solid-state NMR study of a membrane protein selectively labeled through the use of cell-free expression. The sample consists of MscL (mechano-sensitive channel of large conductance), a 75 kDa pentameric a-helical ion channel from Escherichia coli, reconstituted in a hydrated lipid bilayer. Compared to a uniformly labeled protein sample, the spectral crowding is greatly reduced in the cell-free expressed protein sample. This approach may be a decisive step required for spectral assignment and structure determination of membrane proteins by solid-state NMR

NOEnet–Use of NOE networks for NMR resonance assignment of proteins with known 3D structure

Motivation: A prerequisite for any protein study by NMR is the assignment of the resonances from the 15N−1H HSQC spectrum to their corresponding atoms of the protein backbone. Usually, this assignment is obtained by analyzing triple resonance NMR experiments. An alternative assignment strategy exploits the information given by an already available 3D structure of the same or a homologous protein. Up to now, the algorithms that have been developed around the structure-based assignment strategy have the important drawbacks that they cannot guarantee a high assignment accuracy near to 100%

Double sampling of a faecal immunochemical test is not superior to single sampling for detection of colorectal neoplasia: a colonoscopy controlled prospective cohort study

<p>Abstract</p> <p>Background</p> <p>A single sampled faecal immunochemical test (FIT) has moderate sensitivity for colorectal cancer and advanced adenomas. Repeated FIT sampling could improve test sensitivity. The aim of the present study is to determine whether any of three different strategies of double FIT sampling has a better combination of sensitivity and specificity than single FIT sampling.</p> <p>Methods</p> <p>Test performance of single FIT sampling in subjects scheduled for colonoscopy was compared to double FIT sampling intra-individually. Test positivity of double FIT sampling was evaluated in three different ways: 1) "one of two FITs+" when at least one out of two measurements exceeded the cut-off value, 2) "two of two FITs+" when both measurements exceeded the cut-off value, 3) "mean of two FITs+" when the geometric mean of two FITs exceeded the cut-off value. Receiver operator curves were calculated and sensitivity of single and the three strategies of double FIT sampling were compared at a fixed level of specificity.</p> <p>Results</p> <p>In 124 of 1096 subjects, screen relevant neoplasia (SRN) were found (i.e. early stage CRC or advanced adenomas). At any cut-off, "two of two FITs+" resulted in the lowest and "one of two FITs+" in the highest sensitivity for SRN (range 35-44% and 42%-54% respectively). ROC's of double FIT sampling were similar to single FIT sampling. At specificities of 85/90/95%, sensitivity of any double FIT sampling strategy did not differ significantly from single FIT (p-values 0.07-1).</p> <p>Conclusion</p> <p>At any cut off, "one of two FITs+" is the most sensitive double FIT sampling strategy. However, at a given specificity level, sensitivity of any double FIT sampling strategy for SRN is comparable to single FIT sampling at a different cut-off value. None of the double FIT strategies has a superior combination of sensitivity and specificity over single FIT.</p

Robust structure-based resonance assignment for functional protein studies by NMR

High-throughput functional protein NMR studies, like protein interactions or dynamics, require an automated approach for the assignment of the protein backbone. With the availability of a growing number of protein 3D structures, a new class of automated approaches, called structure-based assignment, has been developed quite recently. Structure-based approaches use primarily NMR input data that are not based on J-coupling and for which connections between residues are not limited by through bonds magnetization transfer efficiency. We present here a robust structure-based assignment approach using mainly HN–HN NOEs networks, as well as 1H–15N residual dipolar couplings and chemical shifts. The NOEnet complete search algorithm is robust against assignment errors, even for sparse input data. Instead of a unique and partly erroneous assignment solution, an optimal assignment ensemble with an accuracy equal or near to 100% is given by NOEnet. We show that even low precision assignment ensembles give enough information for functional studies, like modeling of protein-complexes. Finally, the combination of NOEnet with a low number of ambiguous J-coupling sequential connectivities yields a high precision assignment ensemble. NOEnet will be available under: http://www.icsn.cnrs-gif.fr/download/nmr

Etude par RMN (Résonance magnétique nucléaire) des interactions actine-thymosine beta4

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Etude structurale par RMN d'un complexe ARN protéine impliqué dans la régulation de l'utilisation du site A3 d'épissage du VIH-1

ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Etude par RMN d'un complexe d'embrassement entre l'ARN TAR du VIH-1 et un aptamère ADN (étude structurale de l'inhibition de l'utilisation du site A3 d'épissage du VIH-1)

Cette thèse présente l'étude structurale par spectroscopie RMN d'acides nucléiques liés à la régulation du cycle cellulaire du virus VIH-1. La première partie concerne la détermination de la structure d'un complexe d'embrassement entre l'ARN TAR du VIH-1 et un ADN aptamère sélectionné contre cette séquence. La seconde partie est liée à l'étude de l'inhibition de l'utilisation du site A3 d'épissage du VIH-1. La structure de plusieurs ARN contenant cette séquence inhibitrice, nommée ESS2, ainsi que leurs interactions avec la protéine régulatrice UP1, ont été étudiées.This manuscript reveals insights provided by the use of NMR spectroscopy to struqtural studies of nucleic acids involved into HIV-1 life cycle. The first part deals with the structure determination of a kissing complex between HIV-1 TAR RNA and an aptamer DNA selected against it. The second part is related to the structural study of splicing inhibition at A3 site in HIV-1. Several RNA, containing the inhibitory sequence, called ESS2, have been studied by NMR, as also their interactions with the regulating protein UP1.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

ETUDE PAR RMN DES INTERACTIONS ENTRE PROTEINES ET PARTENAIRES BIOLOGIQUES (DETERMINATION DE LA STRUCTURE DES COMPLEXES ALCR-ADN ET CAPSICEINE-STEROL ET ETUDE DE R1B, UN MOTIF DE LIAISON A L'ARN)

ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Méthodologies de criblages d'interactions protéines-ligands par RMN (inhibitions de la Glms et de Bcl-xL)

La versatilité de la Résonance Magnétique Nucléaire (RMN) lui permet des applications dans des domaines variés. Cette versatilité en fait un instrument de première importance dans la recherche de traitements thérapeutiques. Elle permet de déterminer la structure et la dynamique des molécules en interactions. Nous avons utilisé la RMN sur deux protéines impliquées dans diverses pathologies : Bcl-xL, partiellement responsable de la dé cience en apoptose dans certains cancers, et la Glms, connnue pour provoquer des complications chez des patients atteints de diabète de type II et cible dans la lutte anti-bactérienne. Le but était d améliorer notre compréhension des interactions entre ces protéines et de nouveaux ligands capables d inhiber leurs activités. Ceux-ci sont soit extraits de plantes, dans le cas de Bcl-xL, soit de synthèse, dans le cas de Glms. Nos résultats ont permis de donner des orientations quant à l amélioration du pouvoir thérapeutique des ligands étudiés.The versatility of Nuclear Magnetic Resonance (NMR) allows several applications in various domains. This versatility makes it a tool of prime importance in the eld of therapeutic treatments research. It allows the determination of the structure and the dynamic of the interacting molecules. We used NMR on two proteins involved in diverse pathologies : Bcl-xL, partially responsible for the apoptosis de ciency for certain cancers, and the Glms, known to give complications to people affected with type II diabetes and target in the anti-microbial ght. The goal was to enhance our understanding of the interactions between those proteins and new molecules able to inhibit their activities. Those molecules are either extract from plants (Bcl-xL study), or synthesized (Glms study). Our results allowed to give orientations about the enhancements of the therapeutic effects of the studied molecules.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF