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The finite size effects and two-state paradigm of protein folding
Artem Badasyan, Matjaž Valant, Jože Grdadolnik, Vladimir N. Uversky, 2021, original scientific article

Abstract: The coil to globule transition of the polypeptide chain is the physical phenomenon behind the folding of globular proteins. Globular proteins with a single domain usually consist of about 30 to 100 amino acid residues, and this finite size extends the transition interval of the coil-globule phase transition. Based on the pedantic derivation of the two-state model, we introduce the number of amino acid residues of a polypeptide chain as a parameter in the expressions for two cooperativity measures and reveal their physical significance. We conclude that the k2 measure, defined as the ratio of van ’t Hoff and calorimetric enthalpy is related to the degeneracy of the denatured state and describes the number of cooperative units involved in the transition; additionally, it is found that the widely discussed k2=1 is just the necessary condition to classify the protein as the two-state folder. We also find that Ωc, a quantity not limited from above and growing with system size, is simply proportional to the square of the transition interval. This fact allows us to perform the classical size scaling analysis of the coil-globule phase transition. Moreover, these two measures are shown to describe different characteristics of protein folding
Keywords: protein folding, two-state model, size scaling, thermodynamic cooperativity
Published in RUNG: 24.02.2021; Views: 2414; Downloads: 69
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Recombinant proteins co-expressed and co-purified in the presence of antibody fragments
Ario De Marco, 2021, independent scientific component part or a chapter in a monograph

Keywords: protein complexes, affinity purification, nanobodies
Published in RUNG: 17.12.2020; Views: 2392; Downloads: 0
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Water reveals non-Arrhenius kinetics in protein folding experiments
Artem Badasyan, 2020, published scientific conference contribution abstract (invited lecture)

Abstract: Statistical theories describe systems in equilibrium, and cannot be used to study kinetics. However, the theo- ries are based on coarse-grained parameters, that include assumptions regarding the underlying kinetics. If such assumptions are incorrect, the theoretical expressions, used to process the experimental data, will not fit. I report on one such case we have met within the application of Zimm-Bragg [1] theory to process folding experiments, and discuss the reasons and consequences. Studies of relaxation phenomena in glass-forming liquids by default account for the shift in temperature by some value, corresponding to the glass formation temperature, .In particular, temperature shift appears in hydrated proteins because of the presence of partially glassy states giving rise to non- Arrhenius relaxation times log τ ~ [2]. A phenomenological approach was suggested by Adam and Gibbs as early as in 1965 to describe the sudden increase of viscosity and the slowing down of the collective modes in super-cooled liquids as the temperature is approaching[3]. The key idea of Adam-Gibbs theory was to consider the supercooled liquid as a set of clusters (cooperatively rearranging regions) of different sizes that change with temperature, giving rise to the shift in re- laxation time. The temperature shift factor is present in many theories describing properties of water. Thus, Truskett and Dill had to include the Adamm-Gibbs temperature shift into their simple analytical model of water to achieve the agreement with experimental data on the tem- perature dependence of self-diffusion coefficient [4]. Later, Schiro and Weik have summarised recent in vitro and in silico experimental results regarding the role of hydration water in the onset of protein structural dy- namics, and have reported the presence of super-Arrhenius relaxation region above the ”protein dynamic transition” temperature [4]. Recently, Mallamace et al have used the Adam-Gibbs theory in their NMR meas- urements of protein folding-unfolding in water [4] and to rationalise the complicated pressure-temperature diagrams in these glass-forming systems. Motivated by the considerations above, and taking into account the relationship between the unimolecular rate of folding in water and the relaxation time 45 , we introduce the tem- perature shift into the formulas used to fit experimental data on hydrated polypeptides. By doing so we resolve the paradox and complete the new method of processing the Circular Dichroism ex- perimental data on protein folding
Keywords: water, protein folding, non-Arrhenius kinetics
Published in RUNG: 20.07.2020; Views: 2960; Downloads: 112
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Pre-immune libraries of recombinant nanobodies: outcome from 15-year experience
Ario De Marco, invited lecture at foreign university

Keywords: Nanobodies, in vitro panning, protein functionalization
Published in RUNG: 14.02.2020; Views: 3062; Downloads: 0
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Quality Control of Purified Proteins to Improve Research Data Reproducibility
Stephan Uebel, Ario De Marco, Nick Berrow, Mario Lebendiker, Maria Garcia-Alai, Stefan Knauer, Blanca Lopez-Mendez, Andrea Matagne, Annabel Parret, Kim Remans, Bertrand Raynal, 2019, published scientific conference contribution abstract (invited lecture)

Keywords: protein quality, biophysical analysis, aggregation
Published in RUNG: 15.01.2020; Views: 2829; Downloads: 0
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Dissecting the role of REEP1 in preventing Tau-mediated neurodegeneration in a D.melanogaster Alzheimer's disease model
Alessio Guglielmi, 2019, doctoral dissertation

Abstract: Tau is natively an unfolded protein that promotes the assembly and the stability of the axonal microtubules in the central nervous system. Increased formation of Tau protein aggregates has been causatively implicated in several neurodegenerative diseases called tauopathies. In the present study, we used the Drosophila melanogaster system to express the longest isoform of human Tau (2N4R) in the nervous system of adult flies, recreating the main features of the human pathology. Herein, this Tau-mediated neurodegeneration model was used as a platform to perform genetic screenings to identify putative modifiers of Tau toxicity. Our strategy exploited the modulation of genes considered as risk factors of Alzheimer’s disease (AD), Frontotemporal Dementias and other neurodegenerative diseases by RNA interference in vivo. This approach allowed us to identify a new gene which participates in the neuronal response against Tau induced neurotoxicity in Drosophila: D-Reep1, homologue of human REEP1 gene (h-Reep1). D-Reep1 knockout flies showed no apparent phenotypes in physiological growing and developmental conditions, however, they showed peculiar sensitivity to stress conditions. In addition, D-Reep1 knockout enhanced the neurodegeneration mediated by Tau expression in Drosophila eyes. On the contrary, the overexpression of UAS-D-Reep1 and UAS-h-Reep1 abolished the typical rough eye phenotype induced by the presence of Tau. The Co-expression of D-Reep1 in Tau backgrounds did not alter the phosphorylation pattern of this protein while, the presence of D-Reep1 seemed to prevent the formation of Tau aggregates in vivo. Thus, the data support the idea that D-Reep1 exerts a protective role on Tau induced toxicity which is independent of its phosphorylation status. In this work, I analysed the mechanisms behind the neuroprotective role of D-Reep1 and, in particular, I found that REEP1 is involved in the regulation of the unfolded protein response (UPR) through the PERK-ATF4 cascade within the ER. By the activation of this pathway, the neurotoxic aggregates of Tau are removed from Drosophila neuronal tissues rescuing the normal characteristics of the affected tissues. Evidences also suggest that the activation of autophagy was behind the removal of Tau aggregates, providing new molecular information about the physiological role of D Reep1 in the nervous system.
Keywords: AD Alzheimer Disease APP Amyloid precursor protein CNS Central Nervous System DM Drosophila melanogaster HSP Hereditary Spastic Paraplegia LN Lewy’s neurite MT Microtubule MAP Microtubule associated protein MT Microtubule/s MTBD Microtubule binding domain NFT Neurofibrillary tangle NP Neuritic plaques PHF Paired helical filament PS1 Presenilin 1 PS2 Presenilin 2 SPG Spastic Paraplegia ThS Thioflavin S
Published in RUNG: 06.12.2019; Views: 3698; Downloads: 122
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