Marco Fioroni

Mechanism by which 2,2,2-trifluoroethanol/water mixtures stabilize secondary-structure formation in peptides: A molecular dynamics study

Molecular dynamics simulation techniques have been used to investigate the effect of 2,2,2-trifluoroethanol (TFE) as a cosolvent on the stability of three different secondary structure-forming peptides: the α-helix from Melittin, the three-stranded β-sheet peptide Betanova, and the β-hairpin 41–56 from the B1 domain of protein G. The peptides were studied in pure water and 30% (vol/vol) TFE/water mixtures at 300 K. The simulations suggest that the stabilizing effect of TFE is induced by the preferential aggregation of TFE molecules around the peptides. This coating displaces water, thereby removing alternative hydrogen-bonding partners and providing a low dielectric environment that favors the formation of intrapeptide hydrogen bonds. Because TFE interacts only weakly with nonpolar residues, hydrophobic interactions within the peptides are not disrupted. As a consequence, TFE promotes stability rather than inducing denaturation.

Directed evolution of a thermophilic endoglucanase (Cel5A) into highly active Cel5A variants with an expanded temperature profile.

Cel5A is a highly active endoglucanase from Thermoanaerobacter tengcongensis MB4, displaying an optimal temperature range between 75 and 80°C. After three rounds of error-prone PCR and screening of 4700 mutants, five variants of Cel5A with improved activities were identified by Congo Red based screening method. Compared with the wild type, the best variants 3F6 and C3-13 display 135±6% and 193±8% of the wild type specific activity for the substrate carboxymethyl cellulose (CMC), besides improvements in the relative expression level in Escherichia coli system. Remarkable are especially the improvements in activities at reduced temperatures (50% of maximum activity at 50°C and about 45°C respectively, while 65°C for the wild type). Molecular Dynamics simulations performed on the 3F6 and C3-13 variants show a decreased number of intra-Cel5A hydrogen bonds compared to the wild type, implying a more flexible protein skeleton which correlates well to the higher catalytic activity at lower temperatures. To investigate functions of each individual amino acid position site-directed (saturation) mutagenesis were generated and screened. Amino acid positions Val249 and Ile321 were found to be crucial for improving activity and residue Ile13 (encoded by rare codon AUA) yields an improved expression level in E. coli.

Effect of hexafluoroisopropanol alcohol on the structure of melittin: A molecular dynamics simulation study

The molecular mechanism by which HFIP stabilizes the α‐helical structure of peptides is not well understood. In the present study, we use melittin as a model to gain insight into the details of the atomistic interactions of HFIP with the peptide. We have performed extensive comparative molecular dynamics simulations (up to 100 nsec) in the absence and in the presence of HFIP. In agreement with recent NMR experiments, the simulations show rapid loss of tertiary structure in water at pH 2 but much higher helicity in 35% HFIP. The MD simulations also indicate that melittin adopts a highly dynamic global structure in 35% HFIP solution with two α‐helical segments sampling a wide range of angular orientations. The analysis of the HFIP distribution shows the tendency of HFIP to aggregate around the peptide, increasing the local cosolvent concentration to more than two times that in the bulk concentration. The correlation of local peptide structure with HFIP coating suggests that displacement of water at the peptide surface is the main contribution of HFIP in stabilizing the secondary structure of melittin. Finally, a stabilizing effect promoted by the presence of counter‐ions was also observed in the simulations.

Functionalized Nanocompartments (Synthosomes) with a Reduction‐Triggered Release System

Biologically derived compartments are constrained in design by their biological functions to ensure life at ambient temperature. Polymer vesicles can be designed to match application demands, such as mechanical stability, organic solvent, substrate and product tolerance, and permeation resistance, that are out of reach for biologically derived vesicles. Synthosomes use, in contrast to polymersomes, a transmembrane channel for controlling the in and out compound fluxes. The block copolymers in synthosomes prevent compound penetration through the polymer shell, whereas polymersomes depend on the diffusion of substrate and product molecules through the polymer shell. The main advantage of synthosomes over polymersomes is that, through protein engineering, it is possible to design functionalized protein channels. A protein channel that can function as an on/off switch offers opportunities for the design of functional nanocompartments with potential applications in synthetic biology (pathway engineering), medicine (drug release), and industrial biotechnology (chiral nanoreactors, multistep syntheses, bioconversions in nonaqueous environments, and selective product recovery). The channel proteins FhuA, OmpF, and Tsx have been incorporated, in functional active form, into blockcopolymer membranes. FhuA, ferric hydroxamate uptake protein component, is a large monomeric transmembrane protein of 714 amino acids folded into 22 antiparallel b strands and made up of two domains. Crystal structures of FhuA have been resolved, and a large passive diffusion channel (FhuA D1–160) was designed by removing a capping globular domain (deletion of amino acids 5–160). FhuA and Tsx were crystallized as monomers and OmpFas a trimer. FhuA and its engineered variants have a significantly wider channel than OmpF (OmpF 27–38 5, FhuA 39–46 5) and this allows even the translocation of single-stranded DNA. The aim and novelty of our work is the introduction of a triggering system, by means of a reduction-triggered “release switch” based on an engineered FhuA channel variant. To the best of our knowledge, in none of the reported triggered systems, was a channel protein employed as a switch. In fact, for polymersomes, a pH trigger, a temperatureassisted pH trigger, and a combined pH/salt trigger have been developed. Furthermore, hydrogen peroxide generation was used for polymer-vesicle degradation by glucose oxidase catalyzing glucose oxidation, and a pH-triggered release system for a polypeptide vesicle has been reported. For synthosomes, the activation of an encapsulated phosphatase after a change in the pH value has been reported. To build up a reduction-triggered release system in synthosomes, the amino-group-labeling agents 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (pyridyl label) and (2-[biotinamido]ethylamido)-3,3’-dithiodipropionic acid N-hydroxysuccinimide ester (biotinyl label) were selected, due to size considerations and the presence of a cleavable disulfide bond within the labeling reagents. Reagents for the specific labeling of amino, hydroxy, carboxyl, and sulfhydryl groups have been well studied and are routinely used for protein modifications. The synthosome calcein release system proposed herein is a triggered release system in which the entrapped compound (calcein) is liberated through an engineered transmembrane channel (FhuA D1–160) upon addition of a reducing agent. Interestingly, label size played an important role in calcein release. A detection protocol for calcein release from liposomes through wild-type FhuA and FhuA D1–160 has been reported. The liposomes were loaded with calcein at a selfquenching concentration (50 mm) and calcein release was achieved by addition of wild-type FhuA and FhuA D1–160. The fluorescence generation upon calcein release was used to record the release kinetics. In order to build a reduction-triggered release system, the amino groups of lysine residues in FhuA D1–160 were modified with either a pyridyl or a biotinyl label (see above). Figure 1 illustrates the reactions for FhuA D1–160 with eight lysine residues (L167, L226, L344, L364, L455, L537, L556, and L586) chemically modified with pyridyl (left) or biotinyl labels (right). Upon disulfide-bond reduction with DTT, a 3-thiopropionic amide group remains on the lysine residues of the FhuA D1–160 with both labels (Figure 1, upper part). Details [*] Dr. O. Onaca, P. Sarkar, Dr. D. Roccatano, A. G ven, Dr. M. Fioroni, Prof. Dr. U. Schwaneberg School of Engineering and Science, Jacobs University Bremen Campus Ring 8, 28759 Bremen (Germany) Fax: (+49)421-200-3543 E-mail: u.schwaneberg@jacobs-university.de

Nanocompartments with a pH release system based on an engineered OmpF channel protein

Synthosomes are a subclass of Polymersomes with a block copolymer membrane (PMOXA–PDMS–PMOXA) and a modified embedded transmembrane channel protein which acts as a selective gate. A synthosome based pH release system functioning increasing the pH from 5 to 7 was developed by introducing six histidine mutations to the OmpF constriction site (OmpF 6His). The pH-dependent compound release (acridine orange, positively charged between 5 ≤ pH ≤ 7) has been found to be tuned by the constriction site electrostatics and size by local structural modifications.

Mechanistic comparison of saccharide depolymerization catalyzed by dicarboxylic acids and glycosidases

Dicarboxylic acids have been identified as promising catalysts for the depolymerization of cellulose and other polysaccharides. It has been suggested that they might act in “biomimetic” analogy to the active site of glycosidases, where also two carboxylic groups are present, and it is assumed that one residue acts as proton donor and the other one as proton acceptor. In the present paper, a series of structurally distinct carboxylic acids were experimentally assessed as catalysts in the hydrolysis of cellobiose under fully identical acidic conditions. The results clearly show a pH-dependent activity profile in bulk aqueous solutions without any evidence for a cooperative mechanism. In contrast, the protein environment at the active site in glycosidases was found to be essential for the cooperative action of the two carboxylic acid groups. A detailed computational chemistry study is presented, focusing on the protein electric potential, as well as on a reduced dielectric constant (e) within the active site, resulting from the limited presence of water. These two aspects alter the pKa of the carboxylic acid groups dramatically, providing the necessary local environment for a cooperative proton donor–acceptor mechanism, which cannot be mimicked by simple diacids in bulk aqueous phase.

Structural and dynamical analysis of an engineered FhuA channel protein embedded into a lipid bilayer or a detergent belt

Engineered channel proteins are promising nano-components with applications in nanodelivery and nanoreactors technology. Because few of the engineered channel proteins have been crystallized, solution studies based on Neutron Scattering, Circular Dichroism and NMR play a major role. Consequently, the understanding of membrane proteins dynamics in water/detergent solutions or when embedded in a lipid membrane, can clarify how the environment affects protein behavior. In this study, molecular dynamics simulations of the FhuA Escherichia coli outer membrane channel protein and its engineered FhuA Δ1-159 variant have been performed in two different environments: a DNPC (1,2-dinervonyl-sn-glycero-3-phosphocholine) lipid bilayer and a water/OES (N-octyl-2-hydroxyethyl sulfoxide) detergent solution. Furthermore the FhuA Δ1-159 variant has been simulated in the open and closed states, the last induced by the presence of six 3-(2-pyridyldithio)-propionic-acid in the channel inner core. Differences in protein structural and dynamical behavior between the two environments have been found. Considering the FhuA protein characterized by an elliptical-cylindrical symmetry: (a) neither variations on the secondary structure nor axial deformation have been observed in any of the systems; (b) the ellipticity of the channel section (open state) and its fluctuations are enhanced in presence of water/OES, while diminished or suppressed in the DNPC bilayer; (c) the insertion of hydrophobic pyridyl groups into the FhuA Δ1-159 channel (closed state) induces a higher ellipticity in water/OES solution, while shifting to a circular section in the DNPC membrane; (d) the cork domain represented by the first 159 amino acids does not play a major role for protein stability.

Exploring the mineralization of hydrophobins at a liquid interface

Hydrophobins, a class of highly surface active amphiphilic proteins, can stabilize various interfaces. When used for emulsion stabilization, it has been recently shown that they are able to induce mineralization, resulting in hollow capsules. We found that not all types of hydrophobins trigger mineralization, and that the morphology of the mineral changes depending on the selected oil. We investigated the formation of hydrophobin films at interfaces by the use of CD spectroscopy. In order to elucidate the structural features that enhance the mineralization property and give a possible explanation for this behavior, we performed MD-simulations of two representative hydrophobins (EAS for class I and HFBII for class II) at a hexane–water interface, in the presence as well as in the absence of ions. Our studies showed that the class II hydrophobin HFBII, which did not induce mineralization, only slightly changes its structure during adsorption at the oil–water interface or upon addition of Ca2+ and HPO42− ions. In contrast to that, the class I hydrophobin EAS changed its conformation to a large extent during the adsorption and interacted strongly with added ions. We revealed that EAS preorganizes the ions at short distances matching the lattice dimensions of hydroxyapatite. The latter finding yields a straightforward explanation for the observed differences in mineralization behavior and allows us to search for other hydrophobins that could assist mineralization, despite their different functions in nature.

Effect of Na+, Mg2+, and Zn2+ chlorides on the structural and thermodynamic properties of water/n‐heptane interfaces

The effect on the structural and thermodynamic properties in water/n‐heptane interfaces on addition of NaCl, MgCl2, and ZnCl2 has been examined through five independent 100‐ns molecular dynamics simulations. Results indicate that the interfacial thickness within the framework of the capillary‐wave model decreases on addition of electrolytes in the order Na+ < Mg2+ < Zn2+, whereas the interfacial tension increases in the same order. Ionic density profiles and self‐diffusion coefficients are strongly influenced by the strength of the first hydration shell, which varies in the order Na+ < Mg2+ < Zn2+. On the other hand, the Cl− behavior, that is, diffusion and solvation sphere, is influenced by its counterion. Accordingly, cations are strongly expelled from the interface, which is especially remarkable for the small divalent cations. This fact alters the water geometry near the interface and in a lesser extent n‐heptane order and number of hydrogen bonds per water molecule close to the interface.

Chiral discrimination in liquid 1,1,1-trifluoropropan-2-ol: A molecular dynamics study

The structural and thermodynamical properties of the R and S enantiomers of 1,1,1trifluoropropan-2-ol (TFIP) have been investigated by molecular-dynamics simulations. In particular, the chiral discrimination (Ch.D.) between the two enantiomers in a racemic solution has been analyzed in detail. Differences in density and enthalpy of vaporization between the pure enantiomeric liquid and the racemic mixture have been found. The comparison of the radial distribution functions and the distribution of the reciprocal orientations of TFIP molecules have shown the presence of a slightly different packing organization in the aforementioned solutions explaining the difference in density and enthalpy of vaporization. Furthermore, the structural analysis of the racemic mixture has shown a strong dependence of the homo- and heterochiral preference by the nature of the functional groups present in the molecule. At 298 K, in the case of CH3, CF3, and hydroxy groups, the homochiral interaction is followed by a heterochira...