NEO-AMYLOPECTINYL MODELS SYNTHESIS OF COMPLEX BETA-BRANCHED MALTO-OLIGOSACCHARIDES IN SOLUBLE AND SOLID PHASE

International audienceStarch is the ubiquitous glucidic reserve compound in nature. Synthesized by most plants from solar energy it is easily produced in very large scale cultures (cereals, tubers, legumes…). Besides its irreplaceable position in the food chain Starch is also a widely used commodity for its non-alimentary properties (paper, textile industry, adhesive, gels…) in almost all human activities. Though the polysaccharidic nature and the basic structure : poly-alpha(1,4) glucopyrannose of its minor constituent Amylose, and alpha-1,6) branched alpha-1,4-polyglucopyrannose of the major amylopectin has long been known, to date, the fine primary structure of the former still remains to be described ! The branching pattern found in amylopectin can reach extreme complexity. The determination of amylopectins' primary structures from various botanical origins families can be a real challenge. Moreover, though the enzymes involved in starch synthesis have been well-described, no satisfactory in-vitro synthesis has been achieved to date, one of the reasons invoked being the lack of proper primer substrate. In this paper we present an investigation of such structural diversity by rebuilding well-defined branched malto-oligosaccharidic model structures through chemical hemisynthesis, in order to obtain such substrates. Several isoamylase resistant, beta-branched neo-amylopectinyl oligosaccharides having degrees of polymerisation (DP) 4 to 8, with well-defined structures, were obtained. The construction of these models was performed using chemically modified malto-oligosaccharides in solution. All structures were confirmed by long distance heteronuclear NMR spectroscopy. Using activated Wang resin, solid-phase supported oligosaccharides were synthesised and the structures analysed by HR-MAS NMR

Structural basis for the sequence-specific RNA-recognition mechanism of human CUG-BP1 RRM3

The CUG-binding protein 1 (CUG-BP1) is a member of the CUG-BP1 and ETR-like factors (CELF) family or the Bruno-like family and is involved in the control of splicing, translation and mRNA degradation. Several target RNA sequences of CUG-BP1 have been predicted, such as the CUG triplet repeat, the GU-rich sequences and the AU-rich element of nuclear pre-mRNAs and/or cytoplasmic mRNA. CUG-BP1 has three RNA-recognition motifs (RRMs), among which the third RRM (RRM3) can bind to the target RNAs on its own. In this study, we solved the solution structure of the CUG-BP1 RRM3 by hetero-nuclear NMR spectroscopy. The CUG-BP1 RRM3 exhibited a noncanonical RRM fold, with the four-stranded b-sheet surface tightly associated with the N-terminal extension. Furthermore, we determined the solution structure of the CUG-BP1 RRM3 in the complex with (UG)3 RNA, and discovered that the UGU trinucleotide is specifically recognized through extensive stacking interactions and hydrogen bonds within the pocket formed by the b-sheet surface and the N-terminal extension. This study revealed the unique mechanism that enables the CUG-BP1 RRM3 to discriminate the short RNA segment from other sequences, thus providing the molecular basis for the comprehension of the role of the RRM3s in the CELF/Bruno-like family

A szekurin-szeparáz rendszer szerkezeti-funkcionális jellemzése = Structural and functional characterisation of the securin-separase system

A munkaterv azt a célt tűzte ki, hogy a sejtciklusban fontos szerepet játszó szeparáz enzim és rendezetlen inhibítora (szekurin) részletes szerkezeti elemzésén keresztül közelebb jutunk a rendezetlen fehérjék funkciójának jobb megértéséhez. Ezen tervek nagyrészt megvalósultak, elsősorban a humán szekurin klónozását, és részletes szerkezeti jellemzését sikerült megoldanunk, ezen eredményeket két cikkben publikáltuk. Elkészültünk többféle szekurin (S. cerevisiae, D. melanogaster és humán) összehasonlító szerkezeti vizsgálatával is, ezen eredmények publikációja folyamatban van. A szeparáz enzim katalitikus doménjének klónozása sikerült, nagyszámú konstrukció készült és több is jól kifejeződő fehérjét eredményezett, amelyek aktivitását azonban nem tudtuk mérni. A munkát folytatjuk, folyamatban van egyrészt a fehérje különböző körülmények közötti kifejezése, denaturációja és renaturációja, és aktív formában való előállítása, másrészt nemzetközi együttműködésben Prof. Darren Hart-tal (EMBL Grenoble) az ESPRIT (Expression of Soluble Proteins by Random Incremental Truncation) könyvtár-szűrési technika alkalmazása oldható és aktivitást mutató szeparáz konstrukció létrehozására. A fehérjék szerkezeti rendezetlenségének vizsgálata eközben számos más területeken is hatékonyan folyt, az OTKA K60694 pályázat támogatásának összesen 19 publikáció jelent meg. | The main goal of the proposed work was the detailed structure-function characterization of separase, a key enzyme in cell-cycle regulation and its intrinsically disordered inhibitor, securin. We planned to clone and isolate the two proteins, characterize their structure and interaction with particular focus on the disordered structural state of securin. We achieved part of this goal, mostly the cloning, expression and structural characterization by NMR of human securin, which was published in two papers. We also completed the comparative structural study of three different securins (S. cerevisiae, D. melanogaster and human), which is under publication. We managed to clone and express several constructs of the catalytic domain of separase, which resulted in soluble forms of the protein, without enzymatic activity, though. We continue this research by varying expression conditions, including denaturation-renaturation cycles to produce active separase, and also by an international collaboration with Prof. Darren Hart-tal (EMBL Grenoble) with the application of the ESPRIT (Expression of Soluble Proteins by Random Incremental Truncation) technique in order to generate enzymatically active separase. Besides studies on the securin-separase system, we have carried out many other lines of productive research on intrinsically disordered proteins with the help of OTKA K60694 grant: we published 19 papers with acknowledging the grant

Rendezetlen fehérjék: a szerkezet-funkció paradigma kiterjesztése = Intrinsically disordered proteins: extension of the structure-function paradigm

Kísérletes eredmények: Rendezetlen chaperonok hatása, a riboszomális fehérjék kettős chaperone hatásának kimutatása. A rendezetlen fehérjék ellenállnak a hideg-denaturációnak. A rendezetlen fehérjéknek egyedi a hidratációja. Rendezetlenség kimutatása liofilizált mintákból. A rendezetlenség korrelál a kromoszómális transzlokációval. Az állványfehérjéknek magas a rendezetlensége. Új NMR technika a rendezetlen fehérjék elemzésére. Az ERD14 NMR jelhozzárendelése és szerkezeti-dinamikai jellemzése. Az androgén receptor rendezetlen régiójának hossza kapcsolatban van a kognitív képességekkel. A rendezetlen fehérjék crowding körülmények között sem tekerednek fel. Elméleti megfigyelések: Kötött állapotban megfigyelhető rendezetlenség (bolyhsosság) elemzése, a domén koncepció kiterjesztése a rendezetlenségre, az amiloidokban mutatkozó rendezetlenség leírása. Bioinformatikai eredmények: A hipertermofil baktériumokban a rendezetlenség aránya alacsony. Az alternatív splicing során kialakuló kettős kódolású szakaszokról átíródó fehérjék rendezetlensége teszi lehetővé az alternatív fehérjék létét. A rendezetlenség közvetlen kapcsolatban van a fehérje funkciójával, így harmadlagos szerkezeti elemnek tekinthető. Predikciós módszer kidolgozása az alternatív splicing során létrejövő fehérje életképességének megállapítására. A poty-vírus VPg sokféle funkcióban vesz részt, köszönhetően szerkezeti flexibilitásának. Egyéb eredmények: Review a rendezetlenség kutatásának új szakaszáról. | Experimental results: Study of the disordered chaperones, the determination of the double chaperone effect of ribosomal proteins, resistance of the IDPs to cold denaturation. Determination of the unique hydration of IDPs. Determination of protein disorder from lyophilized samples. Protein disorder correlates with chromosomal translocation. Scaffold proteins contain a high level of structural disorder. Designing a new NMR method for the study of IDPs. Assignation of NMR signals of ERD14 and its structural-dynamics characterization. The observation that the length of the disordered region of androgen receptor is related to cognitive capabilities. IDPs remain unfolded even under crowding conditions. Theoretical observations: Analysis of disorder in the bound form (fuzziness), extending the domain concept to IDPs, description for disorder in amyloids. Bioniformatical results: Observation of low levels of disorder in hyperthermophil bacteria. Alternative protein forms are able to evolve because of the disordered nature of the transcripts of dual coding sequences, that result from alternative splicing. Disorder is in intimate connection to function, thus it should come under the category of tertiary structural elements. Designing a prediction method to determine the viability of a protein from an alternative splice event. The VPg protein of poty virus possesses many different functions, thanks to its structural flexibility. Other results: Review of the new era of disorder research

NEO-AMYLOPECTINYL MODELS SYNTHESIS OF COMPLEX BETA-BRANCHED MALTO-OLIGOSACCHARIDES IN SOLUBLE AND SOLID PHASE

International audienceStarch is the ubiquitous glucidic reserve compound in nature. Synthesized by most plants from solar energy it is easily produced in very large scale cultures (cereals, tubers, legumes…). Besides its irreplaceable position in the food chain Starch is also a widely used commodity for its non-alimentary properties (paper, textile industry, adhesive, gels…) in almost all human activities. Though the polysaccharidic nature and the basic structure : poly-alpha(1,4) glucopyrannose of its minor constituent Amylose, and alpha-1,6) branched alpha-1,4-polyglucopyrannose of the major amylopectin has long been known, to date, the fine primary structure of the former still remains to be described ! The branching pattern found in amylopectin can reach extreme complexity. The determination of amylopectins' primary structures from various botanical origins families can be a real challenge. Moreover, though the enzymes involved in starch synthesis have been well-described, no satisfactory in-vitro synthesis has been achieved to date, one of the reasons invoked being the lack of proper primer substrate. In this paper we present an investigation of such structural diversity by rebuilding well-defined branched malto-oligosaccharidic model structures through chemical hemisynthesis, in order to obtain such substrates. Several isoamylase resistant, beta-branched neo-amylopectinyl oligosaccharides having degrees of polymerisation (DP) 4 to 8, with well-defined structures, were obtained. The construction of these models was performed using chemically modified malto-oligosaccharides in solution. All structures were confirmed by long distance heteronuclear NMR spectroscopy. Using activated Wang resin, solid-phase supported oligosaccharides were synthesised and the structures analysed by HR-MAS NMR

NMR resonance assignments of NarE, a putative ADP-ribosylating toxin from Neisseria meningitidis

NarE is a 16 kDa protein identified from Neisseria meningitidis, one of the bacterial pathogens responsible for meningitis. NarE belongs to the ADP-ribosyltransferase family and catalyses the transfer of ADP-ribose moieties to arginine residues in target protein acceptors. Many pathogenic bacteria utilize ADP-ribosylating toxins to modify and alter essential functions of eukaryotic cells. NarE was proposed to bind iron through a Fe–S center which is supposed to be implied in catalysis. We have produced and purified uniformly labeled 15N- and 15N/13C-NarE and assigned backbone and side-chain resonances using multidimensional heteronuclear NMR spectroscopy. These assignments provide the starting point for the three-dimensional structure determination of NarE and the characterization of the role of the Fe–S center in the catalytic mechanism