Conformational selection underlies recognition of a molybdoenzyme by its dedicated chaperone

Molecular recognition is central to all biological processes. Understanding the key role played by dedicated chaperones in metalloprotein folding and assembly requires the knowledge of their conformational ensembles. In this study, the NarJ chaperone dedicated to the assembly of the membrane-bound respiratory nitrate reductase complex NarGHI, a molybdenum-iron containing metalloprotein, was taken as a model of dedicated chaperone. The combination of two techniques ie site-directed spin labeling followed by EPR spectroscopy and ion mobility mass spectrometry, was used to get information about the structure and conformational dynamics of the NarJ chaperone upon binding the N-terminus of the NarG metalloprotein partner. By the study of singly spin-labeled proteins, the E119 residue present in a conserved elongated hydrophobic groove of NarJ was shown to be part of the interaction site. Moreover, doubly spin-labeled proteins studied by pulsed double electron-electron resonance (DEER) spectroscopy revealed a large and composite distribution of inter-label distances that evolves into a single preexisting one upon complex formation. Additionally, ion mobility mass spectrometry experiments fully support these findings by revealing the existence of several conformers in equilibrium through the distinction of different drift time curves and the selection of one of them upon complex formation. Taken together our work provides a detailed view of the structural flexibility of a dedicated chaperone and suggests that the exquisite recognition and binding of the N-terminus of the metalloprotein is governed by a conformational selection mechanism

Confident assignment of intact mass tags to human salivary cystatins using top-down Fourier-transform ion cyclotron resonance mass spectrometry

International audienceA hybrid linear ion-trap Fourier-transform ion cyclotron resonance mass spectrometer was used for top-down characterization of the abundant human salivary Cystatins, including S, S1, S2, SA, SN, C, and D, using collisionally activated dissociation (CAD) after chromatographic purification of the native, disulfide intact proteins from saliva. Post-translational modifications and protein sequence polymorphisms arising from single nucleotide polymorphisms (SNPs) were assigned from precursor and product ion masses at a tolerance of 10 ppm allowing confident identification of individual intact mass tags. Cystatins S, S1, S2, SA and SN were cleaved of a N-terminal 20 amino-acid signal peptide, and Cystatin C a 26-residue peptide, to yield a generally conserved N-terminus. In contrast, Cystatin D isoforms with 24 and 28 amino-acid residue N-terminal truncations were found such that their N-termini were not conserved. Cystatin S1 was phosphorylated at Ser3, while S2 was phosphorylated at Ser1 and Ser3 of the mature protein, in agreement with previous work. Both Cystatin D isoforms carried the polymorphism C46R (SNP: rs1799841). The 14328 Da isoform of Cystatin SN previously assigned with polymorphism P31L due to a SNP (rs2070856) was found only in whole saliva. Parotid secretions contained no detectable Cystatins while whole saliva largely mirrored the contents of submandibular/sublingual (SMSL) secretions. Top-down high-resolution mass spectrometry is a powerful tool for the identification and characterization of potential protein biomarkers in saliva

A new boronate ester-based crosslinking strategy allows the design of nonswelling and long-term stable dynamic covalent hydrogels

International audienceTesting libraries of phenylboronic acid derivatives and diols revealed a new crosslinking couple for the formation of viscoelastic hydrogels with tunable properties and long-term stability

Caractérisation Rhéologique d'Hydrogels Covalents Dynamiques

International audienceRésumé : Les hydrogels dynamiques sont des matériaux viscoélastiques, formés via des réactions de réticulation covalente dynamique. Ils présentent des propriétés auto-cicatrisantes, malléables et injectables, ce qui les rend particulièrement intéressants pour diverses applications biomédicales. Cependant, les hydrogels covalents dynamiques ont tendance à gonfler et manquent souvent de stabilité. Pour des applications biomédicales, il est en outre important de respecter des conditions de pH et de température physiologiques. Dans cette étude, des hydrogels à base d'esters de boronate, résultant de la réaction covalente dynamique entre un dérivé de l'acide phénylboronique (PBA) et une variété de diols, en utilisant l'acide hyaluronique comme polymère d'intérêt, ont été synthétisés. La combinaison du PBA de type Wulff (wPBA) et de la glucamine s'est révélée être un couple unique permettant d'obtenir des hydrogels peu gonflants, stables à long terme et cytocompatibles. L’utilisation d’acide hyaluronique de différents poids moléculaires, à des concentrations différentes a permis d’obtenir des hydrogels présentant des comportements macroscopiques très variables en termes de texture. Leurs propriétés rhéologiques ont été caractérisées par l’établissement de spectres mécaniques et par des tests de relaxation de contrainte, montrant des comportements également différents. Les valeurs de temps de relaxation obtenus via les balayages en fréquence et les tests de relaxation de contrainte ont été comparées. Les propriétés de cicatrisation ont également été mises en évidence. L’ensemble des résultats confirme ainsi l’intérêt de ces hydrogels comme support pour des applications biomédicale

Enzymology, structure, and dynamics of acetohydroxy acid isomeroreductase

Acetohydroxy acid isomeroreductase is a key enzyme involved in the biosynthetic pathway of the amino acids isoleucine, valine, and leucine. This enzyme is of great interest in agrochemical research because it is present only in plants and microorganisms, making it a potential target for specific herbicides and fungicides. Moreover, it catalyzes an unusual two-step reaction that is of great fundamental interest. With a view to characterizing both the mechanism of inhibition by potential herbicides and the complex reaction mechanism, various techniques of enzymology, molecular biology, mass spectrometry, X-ray crystallography, and theoretical simulation have been used. The results and conclusions of these studies are described briefly in this paper