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The structure of tetrameric mammalian acylaminoacyl peptidase – a key upstream regulator of the proteasome – was determined by cryo-EM (and elucidated by MD), showing a “shutters-and-channels” substrate selection apparatus created by oligomerization.

Carotenoids contribute to a variety of physiological processes in plants, functioning also as biosynthesis precursors of ABA and strigolactones (SLs). SL biosynthesis starts with the enzymatic conversion of all-trans-β-carotene to 9-cis-β-carotene by the DWARF27 (D27) isomerase. In Arabidopsis, D27 has two closely related paralogs, D27-LIKE1 and D27-LIKE2, which were predicted to be β-carotene-isomerases. In the present study, we characterised D27-LIKE1 and identified some key aspects of its physiological and enzymatic functions in Arabidopsis. d27-like1-1 mutant does not display any strigolactone-deficient traits and exhibits a substantially higher 9-cis-violaxanthin content, which is accompanied by a slightly higher ABA level. In vitro feeding assays with recombinant D27-LIKE1 revealed that the protein exhibits affinity to all β-carotene isoforms but with an exclusive preference towards trans/cis conversions and the interconversion between 9-cis, 13-cis and 15-cis-β-carotene forms, and accepts zeaxanthin and violaxanthin as substrates. Finally, we present evidence showing that D27-LIKE1 mRNA is phloem mobile and D27-LIKE1 is an ancient isomerase with a long evolutionary history. In summary, we demonstrate that D27-LIKE1 is a carotenoid isomerase with multi-substrate specificity and has a characteristic preference towards the catalysation of cis/cis interconversion of carotenoids. Therefore, D27-LIKE1 is a potential regulator of carotenoid cis pools and, eventually, SL and ABA biosynthesis pathways.

The structure of porcine AAP (pAAP) in a covalently bound complex with meropenem was determined by cryo-EM to 2.1 Å resolution, showing the mammalian serine-protease inhibited by a carbapenem antibiotic. AAP is a modulator of the ubiquitin-proteasome degradation system and the site of a drug-drug interaction between the widely used antipsychotic, valproate and carbapenems. The active form of pAAP - a toroidal tetramer - binds four meropenem molecules covalently linked to the catalytic Ser587 of the serine-protease triad, in an acyl-enzyme state. AAP is hindered from fully processing the antibiotic by the displacement and protonation of His707 of the catalytic triad. We show that AAP is made susceptible to the association by its unusually sheltered active pockets and flexible catalytic triads, while the carbapenems possess sufficiently small substituents on their β-lactam rings to fit into the shallow substrate-specificity pocket of the enzyme.

Summary We developed a cost sensitive isotope labelling procedure using a fed‐batch fermentation method and tested its efficiency producing the 15N‐, 13C‐ and 15N/13C‐labelled variants of an amyloidogenic miniprotein (E5: EEEAVRLYIQWLKEGGPSSGRPPPS). E5 is a surface active protein, which forms amyloids in solution. Here, we confirm, using both PM‐IRRAS and AFM measurements, that the air–water interface triggers structural rearrangement and promotes the amyloid formation of E5, and thus it is a suitable test protein to work out efficient isotope labelling schemes even for such difficult sequences. E. coli cells expressing the recombinant, ubiquitin‐fused miniprotein were grown in minimal media containing either unlabelled nutrients, or 15N‐NH4Cl and/or 13C‐D‐Glc. The consumption rates of NH4Cl and D‐Glc were quantitatively monitored during fermentation and their ratio was established to be 1:5 (for NH4Cl: D‐Glc). One‐ and two‐step feeding schemes were custom‐optimized to enhance isotope incorporation expressing five different E5 miniprotein variants. With the currently optimized protocols we could achieve a 1.5‐ to 5‐fold increase of yields of several miniproteins coupled to a similar magnitude of cost reduction as compared to flask labelling protocols., We developed a cost sensitive isotope labelling procedure using a fed‐batch fermentation method and tested its efficiency producing the 15N‐, 13C‐ and 15N/13C‐labelled variants of an amyloidogenic miniprotein E5, which is a surface active and forms amyloids in solution. With the currently optimized protocols we could achieve a 1.5‐ to 5‐fold increase of yields of several miniproteins coupled to a similar magnitude of cost reduction as compared to flask labelling protocols.

Carotenoids are colourful isoprenoids that contribute to a variety of physiological processes in plants. They also function as biosynthesis precursors of abscisic acid (ABA) and strigolactones (SLs). SL biosynthesis starts with the enzymatic conversion of all-trans-β-carotene to 9-cis-β-carotene by the DWARF27 (D27) isomerase. In Arabidopsis, D27 has two closely related paralogs, D27-LIKE1 and D27-LIKE2 which were predicted to be β-carotene-isomerases. Here we characterised D27-LIKE1 and identified some key aspects of its function. Arabidopsis d27-like1-1 mutant does not display any SL or karrikin-deficient traits, however, it exhibits a substantially higher 9-cis-violaxanthin content. In vitro feeding assays with recombinant D27-LIKE1 revealed that the protein exhibits affinity to all β-carotene isoforms but with an exclusive preference towards trans/cis conversions and the interconversion between 9-cis, 13-cis and 15-cis-β-carotene forms. Feeding experiments with zeaxanthin and violaxanthin isomers revealed that D27-LIKE1 accepts these xanthophylls as substrates. The remarkably higher 9-cis-violaxanthin content of the mutant is accompanied by a slightly higher ABA level. Finally, we presented evidence that D27-LIKE1 mRNA is phloem mobile and D27-LIKE1 is an ancient isomerase with long evolutionary history. In summary, we demonstrated that D27-LIKE1 is a carotenoid isomerase with multi-substrate specificity and has a characteristic preference towards the catalysation of cis/cis interconversion of carotenoids. Therefore, D27-LIKE1 is a potential regulator of carotenoid cis pools and eventually, SL and ABA biosynthesis pathways.

The extracellular domain of the glucagon-like peptide-1 receptor, GLP-1R, is responsible for the binding of GLP-1, and a handful of additional agonists (such as exenatide, lixisenatide, and liraglutide) used daily for treating type II diabetes mellitus. Lead discovery and optimization, however, require binding studies, which, in turn, necessitate the total synthesis of GLP-1R, comprising 108 residues. A protein domain of 10–15 kDa size could be obtained either by expression in E. coli or by ligating solid-phase peptide synthesis (SPPS)-made fragments. However, direct overexpression fails to give a properly folded protein, as GLP-1R forms an inclusion body, which fails to refold due to improper disulfide pairing. Several bacterial strains, constructs, and fusion partners were probed and it was found that only co-expression with MBP gave a 3D-fold allowing the native disulfide bond pattern formation. Some fusion partners can act as covalently linked or in situ chaperones for guiding the refolding of GLP-1R toward success. Therefore, the bottleneck to preparing GPCR extracellular domains is the correct pairing of the Cys residues. As a proof-of-concept model, nGLP1-R was made by SPPS to form the purified full-length polypeptide chain, subjected to self-guided or spontaneous Cys pairing. However, the formation of correct SS-pairs was lagging behind any protocol in use support, and the bottleneck of large-scale protein production relies on the risky step of proper refolding, which is sometimes possible only if a suitable fusion partner effectively helps and catalysis of the correct disulfide formation.

By using two different synthetic techniques several polypeptides interacting with Class B type G-protein coupled receptors were prepared. These polypeptides of different lengths (20 ≤ amino acids ≤ 40), structural and aggregation properties, were prepared both by solid phase peptide synthesis (SPPS) and E.coli bacterial expression. Their purity, synthetic yields, by-products and (15)N/(13)Clabelling characteristics were compared as function of i) the applied method, ii) amino acid length and iii) folding propensities. Their tentative yields, costs and "environmental footprints" were analyzed and found as follows. For unlabelled and short polypeptides (n= 20 aa.) the method of choice is the less environmentally friendly however, quick and effective SPPS. If the polypeptide is (un)folded and/or has no aggregation propensity, then SPPS gives relatively good yield (e.g. 14 ± 4%) and a pure product (>97%). For aggregating polypeptides production yields drop for both methods 4 ± 2% (SPPS) and 2 ± 1% (E. coli), respectively. For longer (n≥ 30 aa.) macromolecules (e.g. miniproteins) bacterial expression efficacy gets higher. Moreover biotechnology is "greener", the resulting in raw material is purer (2.8 ± 1.5 mg). All these advantages for at a lower cost: ~4 €/aa. If isotopic labelling is needed for heteronuclear NMR measurements, bacterial expression is the sole option, due to the high cost of (15)N/(13)C labelled Fmoc(Boc)-L-aa-OH starting materials needed for SPPS. In E.coli uniformly double-labelled, pure polypeptides can be obtained for less than 5-700 €/mg, regardless of the length of the polypeptide chain. Thus, chemists are encouraged to use E.coli expression systems when adequate to make not only proteins but polypeptides and miniproteins as well.

Interallelic interactions of membrane proteins are not taken into account while evaluating the pathogenicity of sequence variants in autosomal recessive disorders. Podocin, a membrane-anchored component of the slit diaphragm, is encoded by NPHS2, the major gene mutated in hereditary podocytopathies. We formerly showed that its R229Q variant is only pathogenic when trans-associated to specific 3′ mutations and suggested the causal role of an abnormal C-terminal dimerization. Here we show by FRET analysis and size exclusion chromatography that podocin oligomerization occurs exclusively through the C-terminal tail (residues 283–382): principally through the first C-terminal helical region (H1, 283–313), which forms a coiled coil as shown by circular dichroism spectroscopy, and through the 332–348 region. We show the principal role of the oligomerization sites in mediating interallelic interactions: while the monomer-forming R286Tfs*17 podocin remains membranous irrespective of the coexpressed podocin variant identity, podocin variants with an intact H1 significantly influence each other's localization (r2 = 0.68, P = 9.2 × 10−32). The dominant negative effect resulting in intracellular retention of the pathogenic F344Lfs*4-R229Q heterooligomer occurs in parallel with a reduction in the FRET efficiency, suggesting the causal role of a conformational rearrangement. On the other hand, oligomerization can also promote the membrane localization: it can prevent the endocytosis of F344Lfs*4 or F344* podocin mutants induced by C-terminal truncation. In conclusion, C-terminal oligomerization of podocin can mediate both a dominant negative effect and interallelic complementation. Interallelic interactions of NPHS2 are not restricted to the R229Q variant and have to be considered in compound heterozygous individuals.

The 20 residue long Trp-cage is the smallest protein known, and thus has been the subject of several in vitro and in silico folding studies. Here, we report the multistate folding scenario of the miniprotein in atomic detail. We detected and characterized different in- termediate states by temperature de- pendent NMR measurements of the 15 N and 13 C/ 15 N labeled protein, both at neutral and acidic pH values. We de- veloped a deconvolution technique to characterize the invisible—fully folded, unfolded and intermediate—fast ex- changing states. Using nonlinear fitting methods we can obtain both the ther- modynamic parameters (DH F-I , Tm F-I , DCp F-I and DH I-U , Tm I-U , DCp I-U ) and the NMR chemical shifts of the con- formers of the multistate unfolding process. During the unfolding of Trp- cage distinct intermediates evolve: a fast-exchanging intermediate is present under neutral conditions, whereas a slow-exchanging intermediate-pair emerges at acidic pH. The fast-ex- changing intermediate has a native-like structure with a short a-helix in the G 11 -G 15 segment, whereas the slow-ex- changing intermediate-pair presents elevated dynamics, with no detectable native-like residue contacts in which the G 11 � P 12 peptide bond has either cis or trans conformation. Heteronuclear relaxation studies combined with MD simulations revealed the source of backbone mobility and the nature of structural rearrangements during these transitions. The ability to detect struc- tural and dynamic information about folding intermediates in vitro provides an excellent opportunity to gain new insights into the energetic aspects of the energy landscape of protein fold- ing. Our new experimental data offer exceptional testing ground for further computational simulations.