Methods to determine slow diffusion coefficients of biomolecules. Applications to Engrailed 2, a partially disordered protein

We present new NMR methods to measure slow translational diffusion coefficients of biomolecules. Like the heteronuclear stimulated echo experiment (XSTE), these new methods rely on the storage of information about spatial localization during the diffusion delay as longitudinal polarization of nuclei with long T-1 such as nitrogen-15. The new BEST-XSTE sequence combines features of Band-selective Excitation Short-Transient (BEST) and XSTE methods. By avoiding the saturation of all protons except those of amide groups, one can increase the sensitivity by 45% in small proteins. The new experiment which combines band-Selective Optimized Flip-Angle Short-Transient with XSTE (SOFAST-XSTE) offers an alternative when very short recovery delays are desired. A modification of the HSQC-edited version of the XSTE experiment offers enhanced sensitivity and access to higher resolution in the indirect dimension. These new methods have been applied to detect changes in diffusion coefficients due to dimerization or proteolysis of Engrailed 2, a partially disordered protein

Designed glycopeptidomimetics disrupt protein−protein interactions mediating amyloid β‑peptide aggregation and restore neuroblastoma cell viability

How anti-Alzheimer’s drug candidates that reduce amyloid 1−42 peptide fibrillization interact with the most neurotoxic species is far from being understood. We report herein the capacity of sugar-based peptidomimetics to inhibit both Aβ1−42 early oligomerization and fibrillization. A wide range of bio- and physicochemical techniques, such as a new capillary electrophoresis method, nuclear magnetic resonance, and surface plasmon resonance, were used to identify how these new molecules can delay the aggregation of Aβ1−42. We demonstrate that these molecules interact with soluble oligomers in order to maintain the presence of nontoxic monomers and to prevent fibrillization. These compounds totally suppress the toxicity of Aβ1−42 toward SH-SY5Y neuroblastoma cells, even at substoichiometric concentrations. Furthermore, demonstration that the best molecule combines hydrophobic moieties, hydrogen bond donors and acceptors, ammonium groups, and a hydrophilic β-sheet breaker element provides valuable insight for the future structure-based design of inhibitors of Aβ1−42 aggregation

Distribution of Pico- and Nanosecond Motions in Disordered Proteins from Nuclear Spin Relaxation

Intrinsically disordered proteins and intrinsically disordered regions (IDRs) are ubiquitous in the eukaryotic proteome. The description and understanding of their conformational properties require the development of new experimental, computational, and theoretical approaches. Here, we use nuclear spin relaxation to investigate the distribution of timescales of motions in an IDR from picoseconds to nanoseconds. Nitrogen-15 relaxation rates have been measured at five magnetic fields, ranging from 9.4 to 23.5 T (400-1000 MHz for protons). This exceptional wealth of data allowed us to map the spectral density function for the motions of backbone NH pairs in the partially disordered transcription factor Engrailed at 11 different frequencies. We introduce an approach called interpretation of motions by a projection onto an array of correlation times (IMPACT), which focuses on an array of six correlation times with intervals that are equidistant on a logarithmic scale between 21 ps and 21 ns. The distribution of motions in Engrailed varies smoothly along the protein sequence and is multimodal for most residues, with a prevalence of motions around 1 ns in the IDR. We show that IMPACT often provides better quantitative agreement with experimental data than conventional model-free or extended model-free analyses with two or three correlation times. We introduce a graphical representation that offers a convenient platform for a qualitative discussion of dynamics. Even when relaxation data are only acquired at three magnetic fields that are readily accessible, the IMPACT analysis gives a satisfactory characterization of spectral density functions, thus opening the way to a broad use of this approach

A Photochromic Azobenzene Peptidomimetic of a β-Turn Model Peptide Structure as a Conformational Switch

The insertion of azobenzene moiety in complex molecular protein or peptide systems can lead to molecular switches to be used to determine kinetics of folding/unfolding properties of secondary structures, such as α-helix, β-turn, or β-hairpin. In fact, in azobenzene, absorption of light induces a reversible trans ↔ cis isomerization, which in turns generates a strain or a structure relaxation in the chain that causes peptide folding/unfolding. In particular azobenzene may permit reversible conformational control of hairpin formation. In the present work a synthetic photochromic azobenzene amino acid derivative was incorporated as a turn element to modify the synthetic peptide [Pro7,Asn8,Thr10]CSF114 previously designed to fold as a type I β-turn structure in biomimetic HFA/water solution. In particular, the P-N-H fragment at positions 7–9, involved in a β-hairpin, was replaced by an azobenzene amino acid derivative (synthesized ad hoc) to investigate if the electronic properties of the novel peptidomimetic analog could induce variations in the isomerization process. The absorption spectra of the azopeptidomimetic analog of the type I β-turn structure and of the azobenzene amino acid as control were measured as a function of the irradiation time exciting into the respective first ππ* and nπ* transition bands. Isomerization of the azopeptidomimetic results strongly favored by exciting into the ππ* transition. Moreover, conformational changes induced by the cis↔ trans azopeptidomimetic switch were investigated by NMR in different solvents

Etude du mécanisme d'action de l'homéoprotéine Engrailed 2 (interaction protéine - protéine avec la protéine à domaine forkhead Foxa2 et interaction protéine - membrane)

L identification et la compréhension des bases d interactions entre protéines ou entre protéines et membranes sont fondamentales pour mieux comprendre les mécanismes impliqués et leurs applications. Les travaux présentés ici s intéressent à l homéoprotéine Engrailed 2 (En2HD) et à son interaction avec deux partenaires différents : le facteur de transcription à domaine forkhead Foxa2 et les bicouches lipidiques. La première partie de cette étude a concerné la détermination structurale partielle de la protéine Foxa2 par résonance magnétique nucléaire, puis l analyse de résultats préliminaires de l interaction entre les deux protéines obtenus par des tests de stabilité ou par une étude par résonance plasmonique de surface. La deuxième partie a permis d identifier le milieu membranaire le plus proche des bicouches lipidiques natives et de réaliser une réattribution partielle de la structure de l homéodomaine dans ce milieu, puis de réaliser une étude comparative des interactions entre l homéodomaine ou l hélice III d Engrailed 2 et différents milieux membranaires (micelles, bicelles) par résonance magnétique nucléaire 1H-15N ou par résonance magnétique nucléaire du phosphore.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

A microplate fluorescence assay for DAPA aminotransferase by detection of the vicinal diamine 7,8-diaminopelargonic acid.

International audience7,8-Diaminopelargonic acid (DAPA) aminotransferase is an enzyme of the biotin biosynthetic pathway which plays an essential role in Mycobacterium tuberculosis virulence. Inhibition of this enzyme is a potential strategy to combat this microorganism, the causative agent of tuberculosis. To identify new inhibitors as potential drugs, a simple enzymatic assay for high throughput screening (HTS) is needed. Several methods for measuring DAPA aminotransferase activity are already available. However, requirements for their implementation for HTS are tedious. We described here a microplate fluorescence assay for DAPA aminotransferase that is simple, cheap and sensitive allowing linear detection of DAPA in the range of 20 nM to 50 μM. The principle of the method is the direct detection in the enzymatic reaction mixture of the vicinal diamine DAPA derivatized with ortho-phthalaldehyde (OPA) and 2-mercaptoethanol (2ME). The assay was validated with the known inhibitor desmethyl-KAPA and adapted to microplate for HTS. The structure of the stable fluorescent adduct formed between a vicinal primary diamine and OPA in the presence of 2ME was characterized by mass spectrometry and NMR spectroscopy

Etude biophysique de peptides amyloïdes en présence de membranes (caractérisation de leurs interactions et détermination de leurs structures)

Le peptide amyloïde IAPP, impliqué dans le diabète de type 2, possède la propriété de s agréger, passant d un état monomérique initial à des fibres amyloïdes matures, via des espèces oligomériques. Ce processus d agrégation, qui se produit au contact de la membrane, a été étudié par fluorescence, microscopie électronique, dichroïsme circulaire et RMN. Tout d abord, l influence du modèle membranaire a été mise en évidence, en termes de forme, taille (micelles, bicelles, SUV, LUV) et composition lipidique (chaînes et têtes différentes) sur les cinétiques d agrégation et de changement conformationnel et sur la morphologie des fibres. Nous avons cherché à comprendre le rôle du cholestérol dans les interactions peptide/membranes, du point de vue du peptide et de la membrane, en utilisant des vésicules contenant entre 0 et 30% de cholestérol. Il a ainsi été observé qu un pourcentage élevé de cholestérol semble accélérer la cinétique d agrégation. De plus, des expériences de RMN liquide ont été réalisées dans le but de déterminer la structure du peptide IAPP en présence de bicelles. Les premiers résultats montrent que l extrémité C terminale ne s insère pas dans la membrane et possède une flexibilité importante. Enfin, le peptide IAPP a également été comparé à un peptide antimicrobien aux propriétés amyloïdes, la dermaseptine S9. Ces travaux indiquent que les mécanismes de fibrillation et de perméabilisation membranaire ne sont pas reliés et que le mode d action de la dermaseptine S9 repose sur la formation de pores transitoires impliquant des espèces oligomériques.The amyloid peptide IAPP, which is implicated in type 2 diabetes mellitus, aggregates from an initial monomeric state to amyloid fibrils, via oligomeric species. Peptide aggregation, which takes place through membrane contact, was studied using fluorescence, electron microscopy, circular dichroism and NMR. The effect of membrane model was highlighted, in terms of shape, size (micelles, bicelles, SUV, LUV) and composition (lipid headgroups and acyl chains), on aggregation kinetics, conformational change kinetics and fibril morphology. Next, we wanted to elucidate the role of cholesterol in peptide/membranes interactions using vesicles composed of 0 to 30% cholesterol. High cholesterol content was shown to increase aggregation kinetics. Furthermore, IAPP in the presence of bicelles was studied by liquid state NMR in order to solve its structure under these conditions. First results indicate that the C terminus does not insert into the membrane and has an important flexibility. Finally, IAPP was compared with an antimicrobial and amyloid-like peptide, dermaseptin S9. This study shows that fibril formation and membrane permeabilisation mechanisms are not linked and that dermaseptin S9 binds to membrane in an aggregated state, maybe leading to the formation of a transient pore.PARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF