Biomedical subjects

Henriette Molinari

Publications and source records attributed to Henriette Molinari.

14 recordsLinked to original sources

PubMed2006

NMR dynamic studies suggest that allosteric activation regulates ligand binding in chicken liver bile acid-binding protein.

Apo chicken liver bile acid-binding protein has been structurally characterized by NMR. The dynamic behavior of the protein in its apo- and holo-forms, complexed with chenodeoxycholate, has been determined via (15)N relaxation and steady state heteronuclear (15)N((1)H) nuclear Overhauser effect measurements. The dynamic parameters were obtained at two pH values (5.6 and 7.0) for the apoprotein and at pH 7.0 for the holoprotein, using the model free approach. Relaxation studies, performed at three different magnetic fields, revealed a substantial conformational flexibility on the microsecond to millisecond time scales, mainly localized in the C-terminal face of the beta-barrel. The observed dynamics are primarily caused by the protonation/deprotonation of a buried histidine residue, His(98), located on this flexible face. A network of polar buried side chains, defining a spine going from the E to J strand, is likely to provide the long range connectivity needed to communicate motion from His(98) to the EF loop region. NMR data are accompanied by molecular dynamics simulations, suggesting that His(98) protonation equilibrium is the triggering event for the modulation of a functionally important motion, i.e. the opening/closing at the protein open end, whereas ligand binding stabilizes one of the preexisting conformations (the open form). The results presented here, complemented with an analysis of proteins belonging to the intracellular lipid-binding protein family, are consistent with a model of allosteric activation governing the binding mechanism. The functional role of this mechanism is thoroughly discussed within the framework of the mechanism for the enterohepatic circulation of bile acids.

Allosteric Regulation↗

PubMed2006

Comparison of bovine and porcine beta-lactoglobulin: a mass spectrometric analysis.

Nano-electrospray-ionization mass spectrometry (nano-ESI-MS) is applied to comparison of bovine and porcine beta-lactoglobulin (BLG and PLG). The conformational and oligomeric properties of the two proteins under different solvent and experimental conditions are analyzed. The pH-dependence of dimerization is described for the pH range 2-11. The results indicate maximal dimer accumulation at pH 6 for BLG and pH 4 for PLG, as well as a lower stability of the PLG dimer at pH 4 compared to BLG at pH 6. Conformational stability appears to be higher for BLG at acidic pH, but higher for PLG at basic pH. The higher stability of BLG at low pH is revealed by means of either chemical or thermal denaturation. Equilibrium folding intermediates of both proteins are detected. Finally, conditions are found that promote dissociation of the BLG dimer at pH 6 into folded monomers.

Animals↗

PubMed2005

Determinants of protein stability and folding: comparative analysis of beta-lactoglobulins and liver basic fatty acid binding protein.

A new energy decomposition approach, aimed at identifying residues playing a folding key role, has been applied here to three homologous proteins, belonging to the calycin superfamily, namely bovine and porcine beta-lactoglobulins and Liver basic fatty acid binding protein, sharing the same beta-barrel fold and different degree of sequence identities. All-atom, explicit solvent molecular dynamics simulations around the native conformation were used to generate, for each of the three proteins, energy maps which were further simplified through eigenvalue decomposition. Analysis of the components of the eigenvector associated with the lowest eigenvalue singled out those residues (hot sites) behaving as strongly interacting and possible nucleation centers. The results fit well with experimental folding data and, especially, with the analysis of side chain-side chain interaction conservation.

Amino Acid Sequence↗

PubMed2005

MM/PBSA analysis of molecular dynamics simulations of bovine beta-lactoglobulin: free energy gradients in conformational transitions?

The pH-driven opening and closure of beta-lactoglobulin EF loop, acting as a lid and closing the internal cavity of the protein, has been studied by molecular dynamics (MD) simulations and free energy calculations based on molecular mechanics/Poisson-Boltzmann (PB) solvent-accessible surface area (MM/PBSA) methodology. The forms above and below the transition pH differ presumably only in the protonation state of residue Glu89. MM/PBSA calculations are able to reproduce qualitatively the thermodynamics of the transition. The analysis of MD simulations using a combination of MM/PBSA methodology and the colony energy approach is able to highlight the driving forces implied in the transition. The analysis suggests that global rearrangements take place before the equilibrium local conformation is reached. This conclusion may bear general relevance to conformational transitions in all lipocalins and proteins in general.

Animals↗

PubMed2005

Porcine beta-lactoglobulin chemical unfolding: identification of a non-native alpha-helical intermediate.

The chemical unfolding behavior of porcine beta-lactoglobulin (PLG) has been followed at pH 2 and 6 in the presence of guanidinium hydrochloride. The PLG unfolding transition, monitored by tryptophan fluorescence, far and near UV circular dichroism and 1D-NMR, can be described by a three-state transition suggesting the presence of at least one intermediate state that appears to display an excess of non-native alpha-helical structures. The thermodynamic parameters, as determined through a global analysis fitting procedure, give estimates of the free energy differences of the transitions connecting the native, the intermediate and the unfolded state: DeltaG(NI) (0) = 2.8 +/- 0.7 kcal mol(-1) (pH 2) and 4.2 +/- 0.5 kcal mol(-1) (pH 6) and DeltaG(NU) (0) = 7.2 +/- 0.6 kcal mol(-1) (pH 2) and 6.9 +/- 0.6 kcal mol(-1) (pH 6). CD unfolding data of the bovine species (BLG) have been collected here under the same experimental conditions of PLG to allow a careful comparison of the two beta-lactoglobulins. Intermediates with different characteristics have been identified for BLG and PLG, and their nature has been discussed on a structural analysis basis. The thermodynamic data reported here for PLG and BLG and the comparative analysis with data reported for equine beta lactoglobulin, show that homologous beta-barrel proteins, belonging to the same family and displaying high sequence identity (52-64%) populate unfolding intermediates to different extents, even though a common tendency to the formation of non-native alpha-helical intermediates, can be envisaged. The present results provide a prerequisite foundation of knowledge for the design and interpretation of future folding kinetic studies.

Animals↗

PubMed2004

Heterologous expression of bovine and porcine beta-lactoglobulins in Pichia pastoris: towards a comparative functional characterisation.

Bovine and porcine beta-lactoglobulins were cloned and expressed in host cells with the aim of developing the tools necessary for their structural, functional and conformational characterisation by NMR techniques. Both lipocalins were expressed in Pichia pastoris, where the use of a constitutive promoter turned out to allow the highest productivity. The yield of recombinant proteins was further improved through multiple integration of the encoding genes and by increasing aeration of the transformed cultures. Both proteins were obtained in the culture medium at the concentration of 200 microg/ml. Recombinant lipocalins were purified by ion-exchange chromatography from the culture medium. A preliminary NMR characterisation showed that both proteins were correctly folded.

Animals↗

PubMed2003

NMR solution structure of viscotoxin C1 from Viscum album species Coloratum ohwi: toward a structure-function analysis of viscotoxins.

The high resolution three-dimensional structure of the newly discovered plant viscotoxin C1, from the Asiatic Viscum album ssp. Coloratum ohwi, has been determined in solution by (1)H NMR spectroscopy at pH 3.6 and 285 K. The viscotoxin C1-fold, consisting of a helix-turn-helix motif and a short stretch of an antiparralel beta-sheet is very similar to that found for the highly similar viscotoxins A2 and A3 and for other related thionins. Different functional properties of members of the thionin family are discussed here in light of the structural and electrostatic properties. Among the very homologous family of alpha- and beta-thionins, known for their antimicrobial activity, the viscotoxin subfamily differs from the other members because of its high toxicity against tumoral cells. Key residues for the modulation of viscotoxin cytotoxicity have been identified on the basis of sequence and structural alignment.

Amino Acid Sequence↗

PubMed2003

NMR studies of protein hydration and TEMPOL accessibility.

Understanding the mechanisms of the interaction between a protein surface and its outer molecular environment is of primary relevance for the rational design of new drugs and engineered proteins. Protein surface accessibility is emerging as a new dimension of Structural Biology, since NMR methods have been developed to follow how molecules, even those different from physiological ligands, preferentially approach specific regions of the protein surface. Hen egg-white lysozyme, a paradigmatic example of the state of the art of protein structure and dynamics, has been selected as a model system to study protein surface accessibility. Bound water and soluble spin-labels have been used to investigate the interaction of this enzyme, both free and bound to the inhibitor (NAG)(3), with its molecular environment. No tightly bound water molecules were found inside the enzyme active site, which, conversely, appeared as the most exposed to visits from the soluble paramagnetic probe TEMPOL. From the presented set of data, an integrated view of lysozyme surface accessibility towards water and TEMPOL molecules is obtained.

Binding Sites↗

PubMed2003

EF loop conformational change triggers ligand binding in beta-lactoglobulins.

Beta-lactoglobulins, belonging to the lipocalin family, are a widely studied group of proteins, characterized by the ability to solubilize and transport hydrophobic ligands, especially fatty acids. Despite many reports, the mechanism of ligand binding and the functional role of these proteins is still unclear, and many contradicting concepts are often encountered in the literature. In the present paper the comparative analysis of the binding properties of beta-lactoglobulins has been performed using sequence-derived information, structure-based electrostatic calculations, docking simulations, and NMR experiments. Our results reveal for the first time the mechanism of beta-lactoglobulin ligand binding, which is completely determined by the opening-closing of EF loop, triggered by Glu89 protonation. The alkaline shift observed for Glu89 pKa in porcine beta-lactoglobulin (pKa 9.7) with respect to the bovine species (pKa 5.5) depends upon the interplay of electrostatic effects of few nearby key residues. Porcine protein is therefore able to bind fatty acids provided that the appropriate pH solution conditions are met (pH > 8.6), where the EF loop conformational change can take place. The unusually high pH of binding detected for porcine beta-lactoglobulin seems to be functional to lipases activity. Theoretical pKa calculations extended to representative beta-lactoglobulins allowed the identification of key residues involved in structurally and functionally important electrostatic interactions. The results presented here provide a strong indication that the described conformational change is a common feature of all beta-lactoglobulins.

Amino Acid Sequence↗

PubMed2003

Amino acid empirical contact energy definitions for fold recognition in the space of contact maps.

BACKGROUND: Contradicting evidence has been presented in the literature concerning the effectiveness of empirical contact energies for fold recognition. Empirical contact energies are calculated on the basis of information available from selected protein structures, with respect to a defined reference state, according to the quasi-chemical approximation. Protein-solvent interactions are estimated from residue solvent accessibility. RESULTS: In the approach presented here, contact energies are derived from the potential of mean force theory, several definitions of contact are examined and their performance in fold recognition is evaluated on sets of decoy structures. The best definition of contact is tested, on a more realistic scenario, on all predictions including sidechains accepted in the CASP4 experiment. In 30 out of 35 cases the native structure is correctly recognized and best predictions are usually found among the 10 lowest energy predictions. CONCLUSION: The definition of contact based on van der Waals radii of alpha carbon and side chain heavy atoms is seen to perform better than other definitions involving only alpha carbons, only beta carbons, all heavy atoms or only backbone atoms. An important prerequisite for the applicability of the approach is that the protein structure under study should not exhibit anomalous solvent accessibility, compared to soluble proteins whose structure is deposited in the Protein Data Bank. The combined evaluation of a solvent accessibility parameter and contact energy allows for an effective gross screening of predictive models.

Amino Acids↗

PubMed2003

Protocol for MM/PBSA molecular dynamics simulations of proteins.

Continuum solvent models have been employed in past years for understanding processes such as protein folding or biomolecular association. In the last decade, several attempts have been made to merge atomic detail molecular dynamics simulations with solvent continuum models. Among continuum models, the Poisson-Boltzmann solvent accessible surface area model is one of the oldest and most fundamental. Notwithstanding its wide usage for simulation of biomolecular electrostatic potential, the Poisson-Boltzmann equation has been very seldom used to obtain solvation forces for molecular dynamics simulation. We propose here a fast and reliable methodology to implement continuum forces in standard molecular mechanics and dynamics algorithms. Results for a totally unrestrained 1 ns molecular dynamics simulation of a small protein are quantitatively similar to results obtained by explicit solvent molecular dynamics simulations.

Computer Simulation↗

PubMed2003

Solution structure of chicken liver basic fatty acid binding protein.

Chicken liver basic fatty acid binding protein (Lb-FABP) belongs to the basic-type fatty acid binding proteins, a novel group of proteins isolated from liver of different non mammalian species whose structure is not known. The structure of Lb-FABP has been solved by (1)H NMR. The overall fold of Lb-FABP, common to the other proteins of the family, consists of ten antiparallel beta-strands organised in two nearly ortogonal beta-sheets with two alpha helices closing the protein cavity where small hydrophobic ligands can be bound. The binding specificity of the protein is not known, however, based on the high sequence and structural similarity with an orthologous protein, ileal lipid binding protein, it is suggested that bile acids may be the putative ligands.

Amino Acid Sequence↗

PubMed2003

Competitive binding of fatty acids and the fluorescent probe 1-8-anilinonaphthalene sulfonate to bovine beta-lactoglobulin.

The use of spectroscopy in the study of fatty acids binding to bovine beta-lactoglobulin (BLG) appears to be a difficult task, as these acid compounds, assumed as the protein natural ligands, do not exhibit favorable optical response such as, for example, absorption or fluorescence. Therefore, the BLG fatty-acid equilibrium has been tackled by exploiting the competition between fatty acids and ANS, a widely used fluorescent hydrophobic probe, whose binding sites on the protein have been characterized recently. Two lifetime decays of the ANS-BLG complex have been found; the longer one has been attributed to the internal binding site and the shorter one to the external site. At increasing fatty acids concentration, the fractional weight associated with ANS bound to the internal site drops, in agreement with a model describing the competition of the dye with fatty acids, whereas the external site occupancy appears to be unaffected by the fatty acids binding to BLG. This model is supported by docking studies. An estimate of the acid-binding affinities for BLG has been obtained by implementing the fitting of the bound ANS intensities with a competitive binding model. A relevant dependence has been found upon the solution pH, in the range from 6 to 8, which correlates with the calyx accessibility modulated by the conformation of the EF loop. Fatty acids with longer aliphatic chains (palmitate and laurate) are found to display larger affinities for the protein and the interaction free energy nicely correlates with the number of contacts inside the protein calyx, in agreement with docking simulations.

Anilino Naphthalenesulfonates↗

PubMed2002

Peptide models of folding initiation sites of bovine beta-lactoglobulin: identification of nativelike hydrophobic interactions involving G and H strands.

In an attempt to characterize the early folding events in bovine beta-lactoglobulin (BLG), a set of peptides, covering the flexible N-terminal region and the stable C-terminus beta-core, was synthesized and analyzed by circular dichroism and by nuclear magnetic resonance in water, trifluoroethanol (TFE), and sodium dodecyl sulfate (SDS) below and above the critical micellar concentration. The role of local and long-range hydrophobic interactions in guiding the folding has been investigated. For the peptide fragment covering the more flexible N-terminal region of BLG (beta-strands A, B), where both theoretical predictions and kinetic refolding experiments suggested the formation of non-native alpha-helix, no native long-range contacts were identified, and a helical secondary structure was stabilized only in the presence of 25 mM SDS. At variance, in 50% (v/v) TFE, native, long-range hydrophobic interactions were observed in the peptide covering the core region comprising G and H beta-strands. The side chains involved in these interactions form a nativelike hydrophobic cluster, thus suggesting that the GH region may act as the folding initiation site for BLG. This result is reinforced by the identification, in the urea denaturated BLG, of residual structure located at the level of the GH interface, as evidenced by NMR analysis. These results, in excellent agreement with kinetic, thermodynamic, and cold denaturation folding data, once more underline the utmost importance of the GH region for the stability and folding of BLG. Severe aggregation effects prevented the structural analysis of the peptide covering the EFGH region, indicating that this larger segment does not represent an independent folding domain and that the terminal alpha-helix is necessary for stabilizing the BLG folding core.

Amino Acid Sequence↗