| 1. |
- Azinas, Stavros, et al.
(författare)
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D-strand perturbation and amyloid propensity in beta-2 microglobulin
- 2011
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Ingår i: The FEBS Journal. - : John Wiley & Sons. - 1742-464X .- 1742-4658. ; 278:13, s. 2349-58
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Tidskriftsartikel (refereegranskat)abstract
- Proteins hosting main β-sheets adopt specific strategies to avoid intermolecular interactions leading to aggregation and amyloid deposition. Human beta-2 microglobulin (β2m) displays a typical immunoglobulin fold and is known to be amyloidogenic in vivo. Upon severe kidney deficiency, β2m accumulates in the bloodstream, triggering, over the years, pathological deposition of large amyloid aggregates in joints and bones. A β-bulge observed on the edge D β-strand of some β2m crystal structures has been suggested to be crucial in protecting the protein from amyloid aggregation. Conversely, a straight D-strand, observed in different crystal structures of monomeric β2m, could promote amyloid aggregation. More recently, the different conformations observed for the β2m D-strand have been interpreted as the result of intrinsic flexibility, rather than being assigned to a functional protective role against aggregation. To shed light on such contrasting picture, the mutation Asp53→Pro was engineered in β2m, aiming to impair the formation of a regular/straight D-strand. Such a mutant was characterized structurally and biophysically by CD, X-ray crystallography and MS, in addition to an assessment of its amyloid aggregation trends in vitro. The results reported in the present study highlight the conformational plasticity of the edge D-strand, and show that even perturbing the D-strand structure through a Pro residue has only marginal effects on protecting β2m from amyloid aggregation in vitro.
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| 2. |
- Dobritzsch, Doreen, 1972-, et al.
(författare)
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Crystal structure of the productive ternary complex of dihydropyrimidine dehydrogenase with NADPH and 5-iodouracil : Implications for mechanism of inhibition and electron transfer
- 2002
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 277:15, s. 13155-13166
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Tidskriftsartikel (refereegranskat)abstract
- Dihydroprymidine dehydrogenase catalyzes the first and rate-limiting step in pyrimidine degradation by converting pyrimidines to the corresponding 5,6- dihydro compounds. The three-dimensional structures of a binary complex with the inhibitor 5-iodouracil and two ternary complexes with NADPH and the inhibitors 5-iodouracil and uracil-4-acetic acid were determined by x-ray crystallography. In the ternary complexes, NADPH is bound in a catalytically competent fashion, with the nicotinamide ring in a position suitable for hydride transfer to FAD. The structures provide a complete picture of the electron transfer chain from NADPH to the substrate, 5-iodouracil, spanning a distance of 56 A and involving FAD, four [Fe-S] clusters, and FMN as cofactors. The crystallographic analysis further reveals that pyrimidine binding triggers a conformational change of a flexible active-site loop in the alpha/beta-barrel domain, resulting in placement of a catalytically crucial cysteine close to the bound substrate. Loop closure requires physiological pH, which is also necessary for correct binding of NADPH. Binding of the voluminous competitive inhibitor uracil-4-acetic acid prevents loop closure due to steric hindrance. The three-dimensional structure of the ternary complex enzyme-NADPH-5-iodouracil supports the proposal that this compound acts as a mechanism-based inhibitor, covalently modifying the active-site residue Cys-671, resulting in S-(hexahydro-2,4-dioxo-5-pyrimidinyl)cysteine.
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| 3. |
- Ricagno, Stefano
(författare)
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Formyl-coenzyme : a transferase, structure and enzymatic mechanism
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background: Formyl-CoA transferase (Frc) is the first enzyme in a two enzyme pathway responsible for oxalate degradation in Oxalobacter formigenes. This bacterium is a constitutive part of human intestinal flora. Its role as an oxalate scavenger is very important, reports have shown a strong connection between the disappearance of 0. formigenes and the appearance of disorders related to oxalate accumulation (e.g. kidney stones, renal failure, cardiac disorders). Frc is a protein of 428 amino acids and belongs to a newly identified third family of CoA transferases where no structural characterisation was previously available. Moreover an enzymatic mechanism has not been proposed for any member of family III of CoA transferases. Results: Frc has been purified and crystallised; subsequently the three dimensional structure of the enzyme was elucidated by X-ray crystallography to 2.2 A resolution. The monomer structure consists of an N-terminal Rossmann fold-like domain, followed by a small domain connected with the N-terminal domain by a long helix. The C-terminal part of Frc is an elongated 70 amino acids loop that interacts with the Rossmann fold-like domain; thus the monomer is shaped as a ring. The homodimer displays a protein fold never observed before, the two subunits are interlocked as two rings of a chain. The structure of Frc in complex with coenzyme A was solved in order to pinpoint the active site. CoA binds to the Rossmann fold-like domain at the nucleotide binding Pap motif; nonetheless CoA binds to it in a very different way. Frc has been characterised kinetically and three mutants of the putative catalytic amino acid Asp169 have been analysed structurally and kinetically. These mutants are almost or totally inactive confirming the importance of Asp169. The structure of Frc in complex with its product oxalyl-CoA has been elucidated. It shows the oxalyl moiety covalently bound to Asp169 as oxalyl-aspartic anhydride. This confirms the existence of anhydrides as intermediates of the reaction and that Asp 169 is the amino acid performing the nucleophilic attack on formyl-CoA.
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| 4. |
- Sala, Benedetta Maria, et al.
(författare)
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Conformational Stability and Dynamics in Crystals Recapitulate Protein Behavior in Solution
- 2020
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Ingår i: Biophysical Journal. - : Ceii Press. - 0006-3495 .- 1542-0086. ; 119:5, s. 978-988
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Tidskriftsartikel (refereegranskat)abstract
- A growing body of evidences has established that in many cases proteins may preserve most of their function and flexibility in a crystalline environment, and several techniques are today capable to characterize molecular properties of proteins in tightly packed lattices. Intriguingly, in the case of amyloidogenic precursors, the presence of transiently populated states (hidden to conventional crystallographic studies) can be correlated to the pathological fate of the native fold; the low fold stability of the native state is a hallmark of aggregation propensity. It remains unclear, however, to which extent biophysical properties of proteins such as the presence of transient conformations or protein stability characterized in crystallo reflect the protein behavior that is more commonly studied in solution. Here, we address this question by investigating some biophysical properties of a prototypical amyloidogenic system, beta 2-microglobulin in solution and in microcrystalline state. By combining NMR chemical shifts with molecular dynamics simulations, we confirmed that conformational dynamics of beta 2-microglobulin native state in the crystal lattice is in keeping with what observed in solution. A comparative study of protein stability in solution and in crystallo is then carried out, monitoring the change in protein secondary structure at increasing temperature by Fourier transform infrared spectroscopy. The increased structural order of the crystalline state contributes to provide better resolved spectral components compared to those collected in solution and crucially, the crystalline samples display thermal stabilities in good agreement with the trend observed in solution. Overall, this workshows that protein stability and occurrence of pathological hidden states in crystals parallel their solution counterpart, confirming the interest of crystals as a platform for the biophysical characterization of processes such as unfolding and aggregation.
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| 5. |
- Visentin, Cristina, et al.
(författare)
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Glycosylation Tunes Neuroserpin Physiological and Pathological Properties
- 2020
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 21:9
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Tidskriftsartikel (refereegranskat)abstract
- Neuroserpin (NS) is a member of the serine protease inhibitors superfamily. Specific point mutations are responsible for its accumulation in the endoplasmic reticulum of neurons that leads to a pathological condition named familial encephalopathy with neuroserpin inclusion bodies (FENIB). Wild-type NS presents two N-glycosylation chains and does not form polymers in vivo, while non-glycosylated NS causes aberrant polymer accumulation in cell models. To date, all in vitro studies have been conducted on bacterially expressed NS, de facto neglecting the role of glycosylation in the biochemical properties of NS. Here, we report the expression and purification of human glycosylated NS (gNS) using a novel eukaryotic expression system, LEXSY. Our results confirm the correct N-glycosylation of wild-type gNS. The fold and stability of gNS are not altered compared to bacterially expressed NS, as demonstrated by the circular dichroism and intrinsic tryptophan fluorescence assays. Intriguingly, gNS displays a remarkably reduced polymerisation propensity compared to non-glycosylated NS, in keeping with what was previously observed for wild-type NS in vivo and in cell models. Thus, our results support the relevance of gNS as a new in vitro tool to study the molecular bases of FENIB.
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