The characterization of Thermotoga maritima Arginine Binding Protein variants demonstrates that minimal local strains have an important impact on protein stability

The Ramachandran plot is a versatile and valuable tool that provides fundamental information for protein structure determination, prediction, and validation. The structural/thermodynamic effects produced by forcing a residue to adopt a conformation predicted to be forbidden were here explored using Thermotoga maritima Arginine Binding Protein (TmArgBP) as model. Specifically, we mutated TmArgBP Gly52 that assumes a conformation believed to be strictly disallowed for non-Gly residues. Surprisingly, the crystallographic characterization of Gly52Ala TmArgBP indicates that the structural context forces the residue to adopt a non-canonical conformation never observed in any of the high-medium resolution PDB structures. Interestingly, the inspection of this high resolution structure demonstrates that only minor alterations occur. Nevertheless, experiments indicate that Gly52 replacements in TmArgBP produce destabilizations comparable to those observed upon protein truncation or dissection in domains. Notably, we show that force-fields commonly used in computational biology do not reproduce this non-canonical state. Using TmArgBP as model system we here demonstrate that the structural context may force residues to adopt conformations believed to be strictly forbidden and that barely detectable alterations produce major destabilizations. Present findings highlight the role of subtle strains in governing protein stability. A full understanding of these phenomena is essential for an exhaustive comprehension of the factors regulating protein structures

Binding of a type 1 RIP and of its chimeric variant to phospholipid bilayers: evidence for a link between cytotoxicity and protein/membrane interactions

Ribosome-inactivating proteins (RIPs) are enzymes, almost all identified in plants, able to kill cells by depurination of rRNAs. Recently, in order to improve resistance to proteolysis of a type 1 RIP (PD-L4), we produced a recombinant chimera combining it with a wheat protease inhibitor (WSCI). Resulting chimeric construct, named PD-L4UWSCI, in addition to present the functions of the two domains, shows also an enhanced cytotoxic action on murine cancer cells when compared to PD-L4. Since different ways of interaction of proteins with membranes imply different resulting effects on cells, in this study we investigate conformational stability of PD-L4 and PD-L4UWSCI and their interaction with membrane models (liposomes). Circular dichroism analysis and differential scanning calorimetry measurements indicate that PD-L4 and PD-L4UWSCI present high and similar conformational stability, whereas analysis of their binding to liposomes, obtained by isothermal titration calorimetry and differential scanning calorimetry, clearly indicate that chimera is able to interact with biomembranes more effectively. Overall, our data point out that WSCI domain, probably because of its flexibility in solution, enhances the chimeric protein interaction with membrane lipid surfaces without however destabilizing the overall protein structure. Analysis of interactions between RIPs or RIP based conjugates and lipid surfaces could provide novel insights in the search of more effective selective membrane therapeutics

Unveiling Molecular Recognition of Sialoglycans by Human Siglec-10

29 p.-6 fig.-2 tab.-7 fig. supl.-2 tab. supl.-1 graph. abst.Siglec-10 is an inhibitory I-type lectin selectively recognizing sialoglycans exposed on cell surfaces, involved in several patho-physiological processes. The key role Siglec-10 plays in the regulation of immune cell functions has made it a potential target for the development of immunotherapeutics against a broad range of diseases. However, the crystal structure of the protein has not been resolved for the time being and the atomic description of Siglec-10 interactions with complex glycans has not been previously unraveled. We present here the first insights of the molecular mechanisms regulating the interaction between Siglec-10 and naturally occurring sialoglycans. We used combined spectroscopic, computational and biophysical approaches to dissect glycans' epitope mapping and conformation upon binding in order to afford a description of the 3D complexes. Our outcomes provide a structural perspective for the rational design and development of high-affinity ligands to control the receptor functionality.This study was supported by the project ‘‘GLYTUNES’’ funded by MIUR Progetti di Ricerca di Rilevante Interesse Nazionale (PRIN 2017) (2017XZ2ZBK, 2019–2022) to A.S.; by progetto POR SATIN and Progetto POR CampaniaOncoterapia to A.M.; by the European Commission (H2020-MSCA- 814102-SWEET CROSSTALK project) to A.M., R.M., and A.S.. This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program under grant agreement No 851356 to R.M. FSE,PON Ricerca e Innovazione 2014–2020, Azione I.1 ‘‘Dottorati Innovativi con caratterizzazione Industriale’’ is acknowledged for funding the PhD grant to R.E.F. Grants by the Spanish Ministry of Science MICINN (CTQ2017-88353-R and fellowship BES 2015–071588 to J.G.-C.) and Wellcome Trust 103744/Z/14/Z to P.R.C. are acknowledged.Peer reviewe

Energetica del riconoscimento del DNA e dell'attivazione trascrizionale del fattore di trascizione tiroideo di ratto (TTF-1)

La principale attività della presente unità è quella di determinare la termodinamica del riconoscimento del DNA e dell’attivazione trascrizionale del fattore di trascrizione tiroideo di ratto (TTF-1). L’Unità prevede di dare una descrizione più completa possibile della stabilità termodinamica della proteina (TTF-1), nonché dell’energetica della sua associazione con il DNA bersaglio e con altre proteine coinvolte nel processo di transattivazione come il fattore di trascrizione Pax-8 sia in forma libera che in complesso con il DNA. La determinazione dei parametri termodinamici dell’associazione tra DNA e proteine o tra proteine, specialmente se in combinazione con informazioni strutturali, è in grado di fornire una completa descrizione dei contributi energetici dell’interazione e aiutare nell’identificazione delle regioni dell’interfaccia di riconoscimento. Tutti i parametri termodinamici dell’associazione, la costante di equilibrio, la variazione dell’energia di Gibbs, dell’entalpia, dell’entropia e della capacità termica, nonchè la stechiometria dell’interazione, saranno determinati mediante misure di calorimetria di titolazione isoterma (ITC) e di fluorescenza. La stabilità conformazionale dell’omeodominio di TTF-1 e di mutanti di amminoacidi presenti nella regione di riconoscimento, saranno studiati mediante dicroismo circolare (CD) e microcalorimetria differenziale a scansione (DSC). Il costrutto contenente il dominio N-terminale + l’omeodominio di TTF-1 sarà studiato mediante CD e DSC per determinare la stabilità termodinamica della proteina in presenza e in assenza del suo DNA bersaglio. Saranno inoltre determinati i parametri termodinamici dell’interazione tra il costrutto contenente il dominio N-terminale + l’omeodominio di TTF-1 e il fattore di trascrizione Pax-8 mediante tecniche calorimetriche e spettroscopiche

Studio termodinamico della stabilità conformazionale di proteine in soluzione: azione di agenti denaturanti e stabilizzanti.

TESI di DOTTORATO in SCIENZE CHIMICH

Structure and function of hemoproteins from cold-adapted organism

Environmental oxygen availability certainly plays a key role in the evolution of polar marine life, as suggested by the physiological and biochemical strategies that the organisms have adopted to acquire, deliver and scavenge oxygen. The psychrophilic Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 gives the opportunity to explore the cellular strategies adopted in vivo by cold-adapted microorganisms to cope with cold and high oxygen concentration. Within vertebrates, the dominant suborder Notothenioidei of the Southern Ocean is one of the most interesting models to study the evolutionary biological responses to extreme environment. Hemoproteins of cold-adapted organisms are likely to fulfil important physiological roles, not only in delivering oxygen to cells, but also in protecting them from the nitrosative and oxidative stress. This thesis will in particular focus on: (i) the structural and functional features of globins of the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125, (ii) the role of neuroglobin (Ngb) recently identified in the brain of Antarctic notothenioid fish. The genome of the cold-adapted bacterium P. haloplanktis TAC125 contains multiple genes encoding three distinct monomeric hemoglobins (Hbs) exhibiting a 2/2 -helical fold (2/2Hb). One of these 2/2Hb (Ph-2/2HbO) has been over-expressed and characterised by spectroscopic analysis, kinetic measurements and computer simulation approaches (Howes et al., 2011; Giordano et al., 2011). The results indicate unique adaptive structural properties, that overall confer higher flexibility to the protein and may facilitate its functioning in the cold by providing greater freedom for the correct positioning of ligand(s). Similar to Ngb, the recombinant protein is hexacoordinated in the ferric and ferrous forms, and shows a strong dependence on pH (Howes et al., 2011; Giordano et al., 2011). Polar fish are a suitable model to learn more about the function of globins in the brain, and especially about their role in species devoid of Hb and Myoglobin (Mb). The finding that Antarctic icefishes retain the Ngb gene despite having lost Hb, and Mb in most species, suggests a crucial function. The function of Ngb needs to be ascertained, because it may have important implications in the physiology and pathology of the brain. The first structural model of fish Ngb was described using molecular dynamics simulations. Specifically, Ngb genes from a colourless-blooded Antarctic icefish species (Chaenocephalus aceratus), and a related red-blooded species (Dissostichus mawsoni), were cloned, the recombinant proteins were expressed and purified, and then sequenced and analysed. Both Antarctic fish Ngbs are hexacoordinated, but have some peculiarities that differentiate them from mammalian counterparts: they have extensions in the N and C termini, interacting with the EF loop, and a gap in the alignment that changes the CD-region structure/dynamics, that has been found to play a key role in human Ngb. The adaptive modifications to compensate for the effects of low temperature appear to primarily rely on a higher flexibility of key parts of the molecular structure and/or decreased overall stability. At all levels analysed, the functionally most crucial adaptation to permanently low temperatures apparently requires molecular flexibility to support cell functioning. Proteins are the major targets for the ensuing mechanisms of adaptation

Interactions with Natural Polyamines and Thermal Stability of DNA. A DSC Study and a Theoretical Reconsideration.

The effects of different amounts of four natural polyamines on the thermodynamics of the thermal denaturation of calf thymus and herring sperm DNA have been studied by means of differential scanning calorimetry. Enthalpy changes and the temperature of the maximum heat effect were determined. The stability of the double helix increases by increasing the polyamine/phosphate ratio and the number of positively charged groups on the polyamine molecule. A combination of Manning's polyelectrolyte theory and McGhee and von Hippel's multiple-site exclusion approach has been demonstrated to give a very good reproducibility of experimental results

The effect of trimethylamine N-oxide on RNase A stability:a DSC study

The thermal stability of bovine pancreatic ribonuclease (RNase A) has been investigated in the presence of trimethylamine N-oxide (TMAO), a naturally occurring osmolyte, by means of differential scanning calorimetry (DSC) and circular dichroism (CD) measurements at neutral and acid pH conditions. It is well known that compatible osmolytes such as TMAO are effective in stabilizing protein structure and counteracting the denaturing effect of urea and GuHCl. Calorimetric results show that TMAO stabilizes RNAse A at pH 7.0 and does not stabilize the protein at pH 4.0. RNase A thermal denaturation in the presence of TMAO is a reversible two-state N ⇄ D process. We also show that TMAO counteracts the urea and guanidinium hydrochloride (GuHCl) denaturing effect at neutral pH, whereas the counteracting ability is lost at acid pH

General Counteraction Exerted by Sugars against Denaturants

The conformational stability of globular proteins is strongly influenced by the addition to water of different co-solutes. Some of the latter destabilize the native state, while others stabilize it. It is emerging that stabilizing agents are able to counteract the action of destabilizing agents. We have already provided experimental evidence that this counteraction is a general phenomenon and offered a rationalization. In the present work, we show that four different sugars, namely fructose, glucose, sucrose, and trehalose, counteract the effect of urea, tetramethylurea, sodium perchlorate, guanidinium chloride, and guanidinium thiocyanate despite the chemical and structural differences of those destabilizing agents. The rationalization we provide is as follows: (a) the solvent-excluded volume effect, a purely entropic effect, stabilizes the native state, whose solvent-accessible surface area is smaller than the one of denatured conformations; (b) the magnitude of the solvent-excluded volume effect increases markedly in ternary solutions because the experimental density of such solutions is larger than that of pure water

Molecular dynamics study of the conformational stability of esterase 2 from Alicyclobacillus acidocaldarius

In this study we investigate the conformational stability of thermophilic esterase 2. MD simulations study allows the identification of two cooperative and coupled domains. Analysis of MD trajectories shows that the last α-helix is the main coupling element