Alberto Barbiroli

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.

DE‐loop mutations affect β2 microglobulin stability, oligomerization, and the low‐pH unfolded form

β2 microglobulin (β2m) is the light chain of class‐I major histocompatibility complex (MHC‐I). Its accumulation in the blood of patients affected by kidney failure leads to amyloid deposition around skeletal joints and bones, a severe condition known as Dialysis Related Amyloidosis (DRA). In an effort to dissect the structural determinants of β2m aggregation, several β2m mutants have been previously studied. Among these, three single‐residue mutations in the loop connecting strands D and E (W60G, W60V, D59P) have been shown to affect β2m amyloidogenic properties, and are here considered. To investigate the biochemical and biophysical properties of wild‐type (w.t.) β2m and the three mutants, we explored thermal unfolding by Trp fluorescence and circular dichroism (CD). The W60G mutant reveals a pronounced increase in conformational stability. Protein oligomerization and reduction kinetics were investigated by electrospray‐ionization mass spectrometry (ESI‐MS). All the mutations analyzed here reduce the protein propensity to form soluble oligomers, suggesting a role for the DE‐loop in intermolecular interactions. A partially folded intermediate, which may be involved in protein aggregation induced by acids, accumulates for all the tested proteins at pH 2.5 under oxidizing conditions. Moreover, the kinetics of disulfide reduction reveals specific differences among the tested mutants. Thus, β2m DE‐loop mutations display long‐range effects, affecting stability and structural properties of the native protein and its low‐pH intermediate. The evidence presented here hints to a crucial role played by the DE‐loop in determining the overall properties of native and partially folded β2m.

Process conditions affect starch structure and its interactions with proteins in rice pasta

Structural changes of starch and proteins in rice pasta were investigated as a function of raw-materials and pasta-making conditions, and their impact on cooking behaviour and glycaemic index was assessed. Rice pasta was prepared from untreated or parboiled rice flour by conventional extrusion or by extrusion-cooking. Starch structure was studied by assessing starch accessibility to specific enzymes (α-amylase and pullulanase), and by evaluating the molecular properties of fragments from enzymatic action. Protein solubility in presence/absence of chaotropes and accessibility of protein cysteine thiols allowed to evaluate the intensity and nature of inter-protein interactions. Parboiling stiffens the protein network in rice flour and makes starch more accessible to hydrolysis. Pasta-making induced further changes in the starch structure, that were most evident in pasta made from untreated rice and were mainly related to the amylopectin fraction. Thus, the interplay among structural modifications on starch and/or proteins affects the features of products.

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.

Unfolding intermediate in the peroxisomal flavoprotein D-amino acid oxidase.

The flavoenzyme d-amino acid oxidase (DAAO) from Rhodotorula gracilis is a peroxisomal enzyme and a prototypical member of the glutathione reductase family of flavoproteins. DAAO is a stable homodimer with a FAD molecule tightly bound to each 40-kDa subunit. In this work, the urea-induced unfolding of dimeric DAAO was compared with that of a monomeric form of the same protein, a deleted dimerization loop mutant. By using circular dichroism spectroscopy, protein and flavin fluorescence, 1,8-anilinonaphtalene sulfonic acid binding and activity assays, we demonstrated that the urea-induced unfolding of DAAO is a three-state process, yielding an intermediate, and that this process is reversible. The intermediate species lacks the catalytic activity and the characteristic tertiary structure of native DAAO but has significant secondary structure and retains flavin binding. Unfolding of DAAO proceeds through formation of an expanded, partially unfolded inactive intermediate, characterized by low solubility, by increased exposure of hydrophobic surfaces, and by increased sensitivity to trypsin of the β-strand F5 belonging to the FAD binding domain. The oligomeric state does not modify the inferred folding process. The strand F5 is in contact with the C-terminal α-helix containing the Ser-Lys-Leu sequence corresponding to the type 1 peroxisomal targeting signal, and this structural element interacts with the N-terminal βαβ flavin binding motif (Rossmann fold). The expanded conformation of the folding intermediate (and in particular the higher disorder of the mentioned secondary structure elements) could match the structure of the inactive holoenzyme required for in vivo trafficking of DAAO through the peroxisomal membrane.

Bovine β-lactoglobulin acts as an acid-resistant drug carrier by exploiting its diverse binding regions

Abstract Binding of fluorine-containing drugs to bovine β-lactoglobulin, the most abundant whey protein in bovine milk, was investigated by means of 19F NMR and mass spectrometry. The stoichiometry of the binding and its stability in acidic medium, where β-lactoglobulin is folded and stable, were also studied, along with competition from molecules that can be regarded as analogs of physiological ligands to bovine β-lactoglobulin. Conditional binding data were combined with protein structural information derived from circular dichroism and limited proteolysis studies. Spectroscopic techniques were also used to assess whether the bound drugs stabilize the protein structure against denaturation by chaotropes or temperature at various pH values. The results obtained provide evidence for the presence of multiple binding regions on the protein, with a specific and different affinity for structurally different classes of hydrophobic drugs and, more generally, that bovine β-lactoglobulin can bind and protect against low pH values various classes of drugs of pharmaceutical relevance.

Prion protein structure is affected by pH-dependent interaction with membranes: a study in a model system.

Interaction of full length recombinant hamster prion protein with liposomes mimicking lipid rafts or non‐raft membrane regions was studied by circular dichroism, chemical cross‐linking and sucrose gradient ultracentrifugation. At pH 7.0, the protein bound palmitoyloleoylphosphatidylcholine/cholesterol/sphingomyelin/monosialoganglioside GM1 (GM1) ganglioside liposomes but not palmitoyloleoylphosphatidylcholine alone (bound/free = 0.33 and 0.01, respectively), maintaining the native α‐helical structure and monomeric form. At pH 5.0, though still binding to quaternary mixtures, in particular GM1, the protein bound also to palmitoyloleoylphosphatidylcholine (bound/free 0.35) becoming unfolded and oligomeric. The pH‐dependent interaction with raft or non‐raft membranes might have implication in vivo, by stabilizing or destabilizing the protein.

One-step purification of Kunitz soybean trypsin inhibitor

A fast and simple method for the extraction and purification of Kunitz trypsin inhibitor from soybean seeds is described. The first step consisted in the heat treatment of whole soybean seeds in water at 60 degrees C for 90 min. It was found that 8.4% of total trypsin inhibitory activity of the seeds was secreted during heat treatment. The aqueous medium was loaded onto an affinity chromatography column with immobilized trypsin. The retained fraction, eluted with 0.01 N HCl, contained the purified Kunitz trypsin inhibitor, which was subsequently stabilized by freeze-drying without loss of activity. From 1g soybean seeds, 0.7 mg inhibitor with a specific trypsin inhibitory (TI) activity of 11,430 TIU/mg was obtained. The yield was greater than that obtained with established procedures. Due to the ease of the procedure proposed, the method is readily scalable to pilot plant or industrial preparations.

Relevance of the flavin binding to the stability and folding of engineered cholesterol oxidase containing noncovalently bound FAD.

The flavoprotein cholesterol oxidase (CO) from Brevibacterium sterolicum is a monomeric flavoenzyme containing one molecule of FAD cofactor covalently linked to His69. The elimination of the covalent link following the His69Ala substitution was demonstrated to result in a significant decrease in activity, in the midpoint redox potential of the flavin, and in stability with respect to the wild‐type enzyme, but does not modify the overall structure of the enzyme. We used CO as a model system to dissect the changes due to the elimination of the covalent link between the flavin and the protein (by comparing the wild‐type and H69A CO holoproteins) with those due to the elimination of the cofactor (by comparing the holo‐ and apoprotein forms of H69A CO). The apoprotein of H69A CO lacks the characteristic tertiary structure of the holoprotein and displays larger hydrophobic surfaces; its urea‐induced unfolding does not occur by a simple two‐state mechanism and is largely nonreversible. Minor alterations in the flavin binding region are evident between the native and the refolded proteins, and are likely responsible for the low refolding yield observed. A model for the equilibrium unfolding of H69A CO that also takes into consideration the effects of cofactor binding and dissociation, and thus may be of general significance in terms of the relationships between cofactor uptake and folding in flavoproteins, is presented.

Crystal structure of LptH, the periplasmic component of the lipopolysaccharide transport machinery from Pseudomonas aeruginosa

Lipopolysaccharide (LPS) is the main glycolipid present in the outer leaflet of the outer membrane (OM) of Gram‐negative bacteria, where it modulates OM permeability, therefore preventing many toxic compounds from entering the cell. LPS biogenesis is an essential process in Gram‐negative bacteria and thus is an ideal target pathway for the development of novel specific antimicrobials. The lipopolysaccharide transport (Lpt) system is responsible for transporting LPS from the periplasmic surface of the inner membrane, where it is assembled, to the cell surface where it is then inserted in the OM. The Lpt system has been widely studied in Escherichia coli, where it consists of seven essential proteins located in the inner membrane (LptBCFG), in the periplasm (LptA) and in the OM (LptDE). In the present study, we focus our attention on the Pseudomonas aeruginosa PAO1 Lpt system. We identified an LptA orthologue, named LptH, and solved its crystal structure at a resolution of 2.75 Å. Using interspecies complementation and site‐directed mutagenesis of a conserved glycine residue, we demonstrate that P. aeruginosa LptH is the genetic and functional homologue of E. coli LptA, with whom it shares the β‐jellyroll fold identified also in other members of the canonical E. coli Lpt model system. Furthermore, we modeled the N‐terminal β‐jellyroll domain of P. aeruginosa LptD, based on the crystal structure of its homologue from Shigella flexneri, aiming to provide more general insight into the mechanism of LPS binding and transport in P. aeruginosa. Both LptH and LptD may represent new targets for the discovery of next generation antibacterial drugs, targeting specific opportunistic pathogens such as P. aeruginosa.