Dimitrios Fessas

DSC studies on bovine serum albumin denaturation Effects of ionic strength and SDS concentration

This work analyzed the thermal denaturation process of defatted bovine serum albumin (BSA). DSC measurements were performed on changing the pH, the ionic strength and the sodium dodecyl sulfate (SDS) concentration. These data have been compared with those previously obtained by us and other authors. The purpose of these measurements was to study the correlation between the three-dimensional organization of BSA native protein structure and its thermodynamic stability and to clarify the non-covalent interactions between the globular proteins and amphipathic molecules. These measurements have shown that the thermal denaturation is always irreversible regardless of pH, ionic strength and SDS concentration. The nature of the irreversible process superimposed on the protein unfolding is discussed. The strong stabilizing effect of NaCl on the BSA native structure has been found for the range 0-1.0 M. It is worth noting that the calorimetric curves, confined to the pH region studied, could not be represented by a two-state transition model; they were deconvoluted as the sum of two independent two-state transitions. These transitions were correlated to the domain structure of BSA. Sodium dodecyl sulfate has a net stabilizing effect up to a molar ratio of 10:1 (ligand to protein). In this range of concentrations the presence of SDS cause a biphasic profile of excess heat capacity. A simple thermodynamic model was developed in attempt to reproduce the experimental DSC profiles and collect information regarding the binding equilibrium of SDS.

Structural basis for dimethylarginine recognition by the Tudor domains of human SMN and SPF30 proteins.

Arginine dimethylation plays critical roles in the assembly of ribonucleoprotein complexes in pre-mRNA splicing and piRNA pathways. We report solution structures of SMN and SPF30 Tudor domains bound to symmetric and asymmetric dimethylated arginine (DMA) that is inherent in the RNP complexes. An aromatic cage in the Tudor domain mediates dimethylarginine recognition by electrostatic stabilization through cation-π interactions. Distinct from extended Tudor domains, dimethylarginine binding by the SMN and SPF30 Tudor domains is independent of proximal residues in the ligand. Yet, enhanced micromolar affinities are obtained by external cooperativity when multiple methylation marks are presented in arginine- and glycine-rich peptide ligands. A hydrogen bond network in the SMN Tudor domain, including Glu134 and a tyrosine hydroxyl of the aromatic cage, enhances cation-π interactions and is impaired by a mutation causing an E134K substitution associated with spinal muscular atrophy. Our structural analysis enables the design of an optimized binding pocket and the prediction of DMA binding properties of Tudor domains.

Water properties in wheat flour dough I: classical thermogravimetry approach

Thermogravimetric analysis allowed inspection of the behavior of water within a wheat flour dough. In such a system water is partitioned between coexisting phases which are none the less far from the true thermodynamic equilibrium. The vaporization rate revealed that water is released in two main steps, the first corresponding to a mere diffusion process, the second being instead related to the desorption of water more tightly bound to the gluten network. It was observed that the overall dough moisture, the extent of mixing, the dough resting time after mixing can modify water partition between phases and the way water is released during the temperature scan. Some effect was also recognized in dough samples to which original water soluble proteins had been added.

Dissecting the structural determinants of the stability of cholesterol oxidase containing covalently bound flavin

Cholesterol oxidase from Brevibacterium sterolicum is a monomeric flavoenzyme catalyzing the oxidation and isomerization of cholesterol to cholest-4-en-3-one. This protein is a class II cholesterol oxidases, with the FAD cofactor covalently linked to the enzyme through the His69 residue. In this work, unfolding of wild-type cholesterol oxidase was compared with that of a H69A mutant, which does not covalently bind the flavin cofactor. The two protein forms do not show significant differences in their overall topology, but the urea-induced unfolding of the H69A mutant occurred at significant lower urea concentrations than wild-type (∼3 versus ∼5 m, respectively), and the mutant protein had a melting temperature ∼10–15 °C lower than wild-type in thermal denaturation experiments. The different sensitivity of the various spectroscopic features used to monitor protein unfolding indicated that in both proteins a two-step (three-state) process occurs. The presence of an intermediate was more evident for the H69A mutant at 2 m urea, where catalytic activity and tertiary structure were lost, and new hydrophobic patches were exposed on the protein surface, resulting in protein aggregation. Comparative analysis of the changes occurring upon urea and thermal treatment of the wild-type and H69A protein showed a good correlation between protein instability and the elimination of the covalent link between the flavin and the protein. This covalent bond represents a structural device to modify the flavin redox potentials and stabilize the tertiary structure of cholesterol oxidase, thus pointing to a specific meaning of the flavin binding mode in enzymes that carry out the same reaction in pathogenic versus non-pathogenic bacteria.

Theseus: A new software package for the handling and analysis of thermal denaturation data of biological macromolecules

A new software package (THESEUS) has been assembled for the analysis of the DSC data, Concerning the thermal denaturation of biological macromolecules. The system is useful to obtain accurate physico-chemical information, bypassing the casual and systematic errors, very common in these experiments. It can also be used for handling data from other instruments and methodologies giving thermodynamic, spectroscopic or other kind of data as a function of temperature. Because many of the researches in this field are of exploratory nature and continuously new unfolding mechanisms are described or hypothesized in the current literature, we have written and assembled this powerful and flexible program of general applicability, in order to put the operator in a position to control each step of the calculation procedure and use his own experience for choosing the better way to solve unexpected problems.ZusammenfassungBei der Analyse von DSC-Daten der thermischen Zersetzung von biologischen Makromolekülen wurde ein neues Softwarepaket (THESEUS) angewendet. Unter Umgehung der in derartigen Experimenten oft vorkommenden gelegentlichen und systematischen Fehler eignet sich dieses System, um genaue Physikalisch-chemische Informationen zu erhalten. Außerdem kann es zur Verarbeitung von Daten verwendet werden, welche von anderen Instrumenten und Methoden stammen, die thermodynamische, spektroskopische oder andere Daten als Funktion der Temperatur liefern. Da viele der Untersuchungen auf diesem Gebiet Forschungs-Charakter tragen und in der gegen-wärtigen Literatur ständig neue Dekonvolutionsmechanismen beschrieben oder angenommen werden, haben wir dieses leistungsstarke, flexible und allgemein anwendbare Programm geschrieben und umgesetzt, um den Operator in die Lage zu versetzen, jeden Phase des Rechenvorganges kontrollieren und seine eigene Erfahrung benutzen zu können, um den besten Weg zur Lösung unvorhergesehener Probleme zu finden.

Starch retrogradation in cooked pasta and rice

ABSTRACT Effect of cooking time on starch retrogradation and water distribution was studied in pasta (spaghetti) and rice (parboiled and arborio) using differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) Optimum cooking times (OCT) were 8, 16, and 18.5 min for spaghetti, parboiled, and arborio rice, respectively. Swelling was observed by image analysis. OCT spaghetti and rice showed various starch retrogradation rates at various aging times and temperatures. Based on the classical Avrami function, the retrogradation rate at 5°C followed the order spaghetti > parboiled rice > arborio rice, while extent was in the opposite order. At higher temperature (20°C), the rates decreased by 20× in all cases. Thermogravimetric analysis (TGA) investigations were undertaken to check the distribution of water within these products and its relationship to starch retrogradation. During heating, water was released in two distinguishable steps at ≈80 and 100°C. Results supported the conclusion that ...

Texture and staling of wheat bread crumb: effects of water extractable proteins and 'pentosans'

The increase of crumb firmness prepared from doughs enriched with water-soluble proteins and/or water-extractable pentosans was investigated at various aging times with DSC, to assess starch retrogradation, and with an Instron dynamometer, to determine the elastic modulus. The crumb enriched in soluble proteins became firmer, and that with extra pentosans remained softer than the standard recipe crumb. It was soon evident that neither starch retrogradation nor water loss (due to the crumb-to-crust migration) were affected by the presence of extra proteins and/or pentosans: thus, neither process could explain the observed differences in the trends of the elastic modulus. According to image analysis investigations, the protein-rich crumb had narrow and regular alveoli, which were well separated from one another, whereas a coarser structure was obtained by adding extra pentosans to the dough recipe. It was, therefore, concluded that differences of crumb firmness might be mainly due to the structure formed in the course of leavening and baking, rather than to starch retrogradation and moisture loss. These processes indeed contribute to significantly increase the rigidity of the alveolar walls, but the overall elastic modulus of a sponge-like system, like a bread crumb, largely depends on the manner in which imposed strains are spread through the alveolar structure. Accordingly, for a given rigidity of the alveolar walls, the elastic modulus of the crumb will depend on the size, shape and distribution of the alveoli.

Starch Gelatinization Kinetics in Bread Dough. DSC investigations on 'simulated' baking processes

Starch gelatinization in wheat flour dough of various moisture contents was quantitatively evaluated by means of DSC. The experimental records were worked out in the form of excess heat capacity vs. T traces which were deconvoluted to single out the contribution of starch gelatinization from that of the decomposition of amylose-lipid complexes. The quantitative procedure used put into evidence that a third endothermic process would take place in the dough with a poor moisture content.DSC runs carried out with sealed pans (i.e., at constant moisture level) and open pans (from which some water was free to evaporate) allow simulation of two extreme conditions of a real baking process, namely that relevant to the central core and to superficial layer of a dough loaf, respectively. The extent of starch gelatinization occurred in these conditions was quantitatively assessed. These data were collected at various heating rates and used to define temperature-time-transformation(TTT) diagrams which are useful tools to predict the progress of baking for any given thermal history of the system.

The X-Ray Structure of Zebrafish (Danio rerio) Ileal Bile Acid-Binding Protein Reveals the Presence of Binding Sites on the Surface of the Protein Molecule

The ileal bile acid-binding proteins (I-BABPs), also called ileal lipid-binding proteins or gastrotropins, belong to the family of the fatty acid-binding proteins and play an important role in the solubilization and transport of bile acids in the enterocyte. This article describes the expression, purification, crystallization, and three-dimensional structure determination of zebrafish (Danio rerio) I-BABP both in its apo form and bound to cholic acid. This is the first X-ray structure of an I-BABP. The structure of the apoprotein was determined to a resolution of 1.6 A, and two different monoclinic crystal forms of the holoprotein were solved and refined to 2.2 A resolution. Three protein molecules are present in the asymmetric unit of one of the co-crystal forms and two in the other, and therefore, the results of this study refer to observations made on five different protein molecules in the crystalline state. In every case, two cholate ligands were found bound in approximately the same position in the internal cavity of the protein molecules, but an unexpected result is the presence of clear and unambiguous electron density for several cholate molecules bound on hydrophobic patches on the surface of all the five independent protein molecules examined. Isothermal titration calorimetry was used for the thermodynamic characterization of the binding mechanism and has yielded results that are consistent with the X-ray data. Ligand binding is described in detail, and the conformational changes undergone by the protein molecule in the apo-to-holo transition are examined by superposition of the apo- and holoprotein models. The structure of the holoprotein is also compared with that of the liver BABP from the same species and those of other I-BABPs determined by NMR.

Contribution of the dimeric state to the thermal stability of the flavoprotein D-amino acid oxidase.

The flavoenzyme DAAO from Rhodotorula gracilis, a structural paradigm of the glutathione‐reductase family of flavoproteins, is a stable homodimer with a flavin adenine dinucleotide (FAD) molecule tightly bound to each 40‐kD subunit. In this work, the thermal unfolding of dimeric DAAO was compared with that of two monomeric forms of the same protein: a Δloop mutant, in which 14 residues belonging to a loop connecting strands βF5–βF6 have been deleted, and a monomer obtained by treating the native holoenzyme with 0.5 M NH4SCN. Thiocyanate specifically and reversibly affects monomer association in wild‐type DAAO by acting on hydrophobic residues and on ionic pairs between the βF5–βF6 loop of one monomer and the αI3′ and αI3″ helices of the symmetry‐related monomer. By using circular dichroism spectroscopy, protein and flavin fluorescence, activity assays, and DSC, we demonstrated that thermal unfolding involves (in order of increasing temperatures) loss of tertiary structure, followed by loss of some elements of secondary structure, and by general unfolding of the protein structure that was concomitant to FAD release. Temperature stability of wild‐type DAAO is related to the presence of a dimeric structure that affects the stability of independent structural domains. The monomeric Δloop mutant is thermodynamically less stable than dimeric wild‐type DAAO (with melting temperatures (Tms) of 48°C and 54°C, respectively). The absence of complications ensuing from association equilibria in the mutant Δloop DAAO allowed identification of two energetic domains: a low‐temperature energetic domain related to unfolding of tertiary structure, and a high‐temperature energetic domain related to loss of secondary structure elements and to flavin release.