Synthèse de nouveaux agents chimiques pour la détermination de structures tridimensionnelles à basse résolution de protéines

La détermination de structures tridimensionnelles des protéines est essentielle à l'étude de leur fonction in vivo. Des stratégies alternatives à l'utilisation de méthodes d'analyse à haute résolution ont été développées dans le but de lever certaines contraintes (e.g. nécessité de quantités de protéines pures cristallisées importantes pour réaliser des expériences de diffraction des rayons X). Ces méthodes sont basées sur l'utilisation conjointe d'agents chimiques de modification de protéines et de l'analyse par spectrométrie de masse. Elles permettent d'obtenir efficacement des données structurales variées (e.g. accessibilité au solvant, contraintes de distances, sites de modifications post-traductionnelles) basse résolution. La difficulté majeure à l'obtention de ces données reste cependant la détection de peptides d'intérêt faiblement représentés dans un mélange complexe. Trois projets ont été initiés durant ces travaux de thèse dans le but d'améliorer cette détection en spectrométrie de masse afin de fournir plus rapidement des données structurales. - Le projet 1 a permis de mettre au point une nouvelle méthode utilisant conjointement un marqueur UV-absorbant dérivé de l'acide -cyano-4-hydroxycinnamique (HCCA) et une matrice MALDI adaptée. Un effet important de discrimination spectrale a été obtenu, permettant la détection de peptides d'intérêt en spectrométrie de masse MALDI-TOF. Plusieurs nouveaux agents chimiques mono et bifonctionnels ont été synthétisés et utilisés pour l'étude d'accessibilité au solvant et pour mesurer des contraintes de distances au sein de protéines modèles. - Lors du projet 2, une nouvelle stratégie d'extraction de peptides d'intérêt issus de la digestion enzymatique d'une protéine immobilisée a été développée. Elle repose sur l'utilisation de supports solides portant différents agents chimiques mono et bifonctionnels. - Le projet 3 a permis de développer de nouveaux agents chimiques possédant un motif UV-absorbant dérivé afin de faciliter la détermination des sites de phosphorylation par analyse spectrométrique de masse de type MALDI-TOFThe three-dimensional structure determination is essential for understanding biological functions of proteins. Alternative strategies to X-Ray crystallography and NMR have been developed to avoid some constraints (e.g. the need of a large quantity of pure crystallized protein). These strategies are based on the use of chemical reagents for protein modification followed by mass spectrometry analysis providing efficient low-resolution protein structure data (e.g. solvent accessibility, distance constraints, position of post-translational modifications). However, the main difficulty to provide these data is the detection of low concentration of peptides of interest in a complex mixture. During this PhD study, three projects have been initiated to improve this detection by mass spectrometry. - Project 1 provided a new method using a light-absorbing label derived of -cyano-4-hydroxycinnamic acid and a suitable MALDI matrix. A significant spectral discrimination effect was obtained, enabling detection of peptides of interest by MALDI-TOF mass spectrometry. New mono and bifunctional chemical reagents were synthesized and used for the study of solvent accessibility and to measure constraint distances in two model proteins. - During the project 2, a new strategy to isolate peptides of interest after enzymatic digestion of a protein captured on resin beads was developped. This method relies on the use of solid supports loaded by different mono and bifunctional chemical reagents. - In the project 3, new chemical reagents bearing a light-absorbing label were synthesized to facilitate determination of phosphorylation sites by MALDI-TOF mass spectrometryMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Chemical cross-linkers for protein structure studies by mass spectrometry

International audienceThe cross-linking approach combined with MS for protein structure determination is one of the most striking examples of multidisciplinary success. Indeed, it has become clear that the bottleneck of the method was the detection and the identification of low-abundance cross-linked peptides in complex mixtures. Sample treatment or chromatography separation partially addresses these issues. However, the main problem comes from over-represented unmodified peptides, which do not yield any structural information. A real breakthrough was provided by high mass accuracy measurement, because of the outstanding technical developments in MS. This improvement greatly simplified the identification of cross-linked peptides, reducing the possible combinations matching with an observed m/z value. In addition, the huge amount of data collected has to be processed with dedicated software whose role is to propose distance constraints or ideally a structural model of the protein. In addition to instrumentation and algorithms efficiency, significant efforts have been made to design new cross-linkers matching all the requirements in terms of reactivity and selectivity but also displaying probes or reactive systems facilitating the isolation, the detection of cross-links, or the interpretation of MS data. These chemical features are reviewed and commented on in the light of the more recent strategies

Designing non-native iron-binding site on a protein cage for biological synthesis of nanoparticles

In biomineralization processes, a supramolecular organic structure is often used as a template for inorganic nanomaterial synthesis. The E2 protein cage derived from Geobacillus stearothermophilus pyruvate dehydrogenase and formed by the self-assembly of 60 subunits, has been functionalized with non-native iron-mineralization capability by incorporating two types of iron-binding peptides. The non-native peptides introduced at the interior surface do not affect the self-assembly of E2 protein subunits. In contrast to the wild-type, the engineered E2 protein cages can serve as size- and shape-constrained reactors for the synthesis of iron nanoparticles. Electrostatic interactions between anionic amino acids and cationic iron molecules drive the formation of iron oxide nanoparticles within the engineered E2 protein cages. The work expands the investigations on nanomaterial biosynthesis using engineered host-guest encapsulation properties of protein cages

Incorporation of graphene quantum dots, iron, and doxorubicin in/on ferritin nanocages for bimodal imaging and drug delivery

Graphene quantum dots (GQDs) have been emerging as next‐generation bioimaging agents because of their intrinsic strong fluorescence, photostability, aqueous stability, biocompatibility, and facile synthesis. In this work, GQDs are encapsulated in ferritin protein nanocages to develop multi‐functional nanoplatforms toward multi‐modal imaging and cancer therapy. Encapsulation of ultra‐small GQDs is expected to reduce their quick excretion from the body and increase their bioimaging efficiency. To expand the functionality of protein nanocages as multi‐modal imaging nanoprobes capable of both fluorescence and magnetic resonance imaging (MRI), GQDs and iron are encapsulated inside the core of AfFtn‐AA (an engineered ferritin nanocage derived from the archaeon Archaeoglobus fulgidus ). The co‐encapsulation is achieved through an iron‐mediated, self‐assembly of ferritin dimers resulting in the formation of GQD–iron complex in the ferritin nanocages ((GQDs/Fe)AA). The (GQDs/Fe)AA shows high relaxivities in MRI and pH‐sensitive fluorescence with strong fluorescence at low pH values and on MDA‐MB‐231 cells. As an imaging agent and a drug nanocarrier, (GQDs/Fe)AA exhibits negligible cytotoxicity on the cells and a high loading capacity (35%) of doxorubicin. Taken together, the (GQDs/Fe)AA shows promising applications in cancer diagnosis and therapy as a pH‐responsive fluorophore, MRI agent, and drug nanocarrier.Accepted versio

Transmission electron microscopy of samples of Ni-NTA-functionalised gold nanoparticles and E2 protein cages.

<p>Ni-NTA-functionalised gold nanoparticles mixed with (A) E2-WT, (B) E2-LH2, (C) E2-LH5, (D) E2-LH6. White circles indicate Ni-NTA-functionalised gold nanoparticles internalised into oligohistidine modified E2 protein cages. The samples were stained with 1% (w/v) phosphotungstic acid. Scale bars are 100 nm.</p

Oligonucleotides for plasmid construction.

<p>Oligonucleotides for plasmid construction.</p

Method for specific internalisation of peptide coated gold nanoparticles into engineered E2 protein cages.

<p>(a) Site-directed mutagenesis of E2 protein cage’s core with oligohistidine sequences (blue) at the RDGE loop (green) on E2 protein subunits; (b) Surface coating of 3.9 nm gold nanoparticles with a self-assembled monolayer made of peptidols and thiolated alkane ethylene glycol (EG) ligands, functionalised with Ni²⁺ nitrilotriacetic moieties (NTA, Ni²⁺); (c) Specific internalisation by affinity binding of Ni-NTA-functionalised peptide coated gold nanoparticles into E2 protein cages presenting oligohistidine sequences.</p

Ni-NTA-functionalised peptide coated gold nanoparticles.

<p>(A) Size distribution of 212 gold nanoparticles with an average diameter of 3.9 ± 0.8 nm. The insert shows a typical electron microscopy image used for size measurement. Immobilisation of Ni-NTA-functionalised peptide coated gold nanoparticles on a hexa-histidine loaded resin. (B) NTA-functionalised SAM coated gold nanoparticles not loaded with Ni²⁺ present no non-specific binding to hexa-histidine resin. (C) 10% (mol:mol) Ni-NTA-functionalised peptide coated gold nanoparticles fully bind to hexa-histidine resin as no free gold nanoparticles are found in the clear supernatant.</p