Danilo Roccatano

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.

Docking of flexible ligands to flexible receptors in solution by molecular dynamics simulation

In this paper, a method of simulating the docking of small flexible ligands to flexible receptors in water is reported. The method is based on molecular dynamics simulations and is an extension of an algorithm previously reported by Di Nola et al. (Di Nola et al., Proteins 1994;19:174–182). The method allows a fast exploration of the receptor surface, using a high temperature of the center of mass translational motion, while the ligand internal motions, the solvent, and the receptor are simulated at room temperature. In addition, the method allows a fast center of mass motion of the ligand, even in solution. The dampening effect of the solvent can be overcome by applying different weights to the interactions between system subsets (solvent, receptor, and ligand). Specific ligand–receptor distances have been used to compare the results of the simulations with the crystal structure. The method is applied, as a test system, to the docking of the phosphocholine to the immunoglobulin McPC603. The results show the similarity of structure between the complex in solution and in the crystal. Proteins 1999;35:153–162.

An extended x‐ray absorption fine structure study of aqueous solutions by employing molecular dynamics simulations

Bromine–oxygen radial distribution functions [g(r)] have been calculated by means of molecular dynamics simulations for aqueous solutions of rubidium bromide, 2‐bromopropane and bromoethane. X‐ray absorption spectra at the bromine K edge have been recorded for these solutions. The water contribution to the extended x‐ray absorption fine structure spectra has been calculated starting from the gBr,O(r) distribution function. Fits of the x‐ray absorption spectra have been performed directly on the raw experimental data, allowing the reliability of the g(r) distribution functions to be verified. The agreement between theoretical and experimental spectra is satisfactory. A procedure to improve model g(r) functions on the basis of the short‐range structural information provided by extended x‐ray absorption fine structure data is proposed.

β‐Hairpin conformation of fibrillogenic peptides: Structure and α‐β transition mechanism revealed by molecular dynamics simulations

Understanding the conformational transitions that trigger the aggregation and amyloidogenesis of otherwise soluble peptides at atomic resolution is of fundamental relevance for the design of effective therapeutic agents against amyloid‐related disorders. In the present study the transition from ideal α‐helical to β‐hairpin conformations is revealed by long timescale molecular dynamics simulations in explicit water solvent, for two well‐known amyloidogenic peptides: the H1 peptide from prion protein and the Aβ(12–28) fragment from the Aβ(1–42) peptide responsible for Alzheimers disease. The simulations highlight the unfolding of α‐helices, followed by the formation of bent conformations and a final convergence to ordered in register β‐hairpin conformations. The β‐hairpins observed, despite different sequences, exhibit a common dynamic behavior and the presence of a peculiar pattern of the hydrophobic side‐chains, in particular in the region of the turns. These observations hint at a possible common aggregation mechanism for the onset of different amyloid diseases and a common mechanism in the transition to the β‐hairpin structures. Furthermore the simulations presented herein evidence the stabilization of the α‐helical conformations induced by the presence of an organic fluorinated cosolvent. The results of MD simulation in 2,2,2‐trifluoroethanol (TFE)/water mixture provide further evidence that the peptide coating effect of TFE molecules is responsible for the stabilization of the soluble helical conformation. Proteins 2004.

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

Molecular Dynamics Simulation of Protein Folding by Essential Dynamics Sampling: Folding Landscape of Horse Heart Cytochrome c

A new method for simulating the folding process of a protein is reported. The method is based on the essential dynamics sampling technique. In essential dynamics sampling, a usual molecular dynamics simulation is performed, but only those steps, not increasing the distance from a target structure, are accepted. The distance is calculated in a configurational subspace defined by a set of generalized coordinates obtained by an essential dynamics analysis of an equilibrated trajectory. The method was applied to the folding process of horse heart cytochrome c, a protein with approximately 3000 degrees of freedom. Starting from structures, with a root-mean-square deviation of approximately 20 A from the crystal structure, the correct folding was obtained, by utilizing only 106 generalized degrees of freedom, chosen among those accounting for the backbone carbon atoms motions, hence not containing any information on the side chains. The folding pathways found are in agreement with experimental data on the same molecule.

Folding and stability of the three-stranded beta-sheet peptide betanova: Insights from molecular dynamics simulations

The dynamics of the three‐stranded β‐sheet peptide Betanova has been studied at four different temperatures (280, 300, 350, and 450 K by molecular dynamics simulation techniques, in explicit water. Two 20‐ns simulations at 280 K indicate that the peptide remains very flexible under “folding” conditions sampling a range of conformations that together satisfy the nuclear magnetic resonance (NMR)‐derived experimental constraints. Two simulations at 300 K (above the experimental folding temperature) of 20 ns each show partial formation of “native”‐like structure, which also satisfies most of the NOE constraints at 280 K. At higher temperature, the presence of compact states, in which a series of hydrophobic contacts remain present, are observed. This is consistent with experimental observations regarding the role of hydrophobic contacts in determining the peptides stability and in initiating the formation of turns and loops. A set of different structures is shown to satisfy NMR‐derived distance restraints and a possible mechanism for the folding of the peptide into the NMR‐determined structure is proposed. Proteins 2002;46:380–392.

Understanding the Interaction of Block Copolymers with DMPC Lipid Bilayer Using Coarse-Grained Molecular Dynamics Simulations

In this paper, we present a computational model of the adsorption and percolation mechanism of poloxamers (poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) triblock copolymers) across a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayer. A coarse-grained model was used to cope with the long time scale of the percolation process. The simulations have provided details of the interaction mechanism of Pluronics with lipid bilayer. In particular, the results have shown that polymer chains containing a PPO block with a length comparable to the DMPC bilayer thickness, such as P85, tends to percolate across the lipid bilayer. On the contrary, Pluronics with a shorter PPO chain, such as L64 and F38, insert partially into the membrane with the PPO block part while the PEO blocks remain in water on one side of the lipid bilayer. The percolation of the polymers into the lipid tail groups reduces the membrane thickness and increases the area per lipid. These effects are more evident for P85 than L64 or F38. Our findings are qualitatively in good agreement with published small-angle X-ray scattering experiments that have evidenced a thinning effect of Pluronics on the lipid bilayer as well as the role of the length of the PPO block on the permeation process of the polymer through the lipid bilayer. Our theoretical results complement the experimental data with a detailed structural and dynamic model of poloxamers at the interface and inside the lipid bilayer.

Sensitive assay for laboratory evolution of hydroxylases toward aromatic and heterocyclic compounds

Powerful directed evolution methods have been developed for tailoring proteins to our needs in industrial applications. Here, the authors report a medium-throughput assay system designed for screening mutant libraries of oxygenases capable of inserting a hydroxyl group into a C-H bond of aromatic or O-heterocyclic compounds and for exploring the substrate profile of oxygenases. The assay system is based on 4-aminoantipyrine (4-AAP), a colorimetric phenol detection reagent. By using 2 detection wavelengths (509 nm and 600 nm), the authors achieved a linear response from 50 to 800 μM phenol and standard deviations below 11% in 96-well plate assays. The monooxygenase P450 BM-3 and its F87A mutant were used as a model system for medium-throughput assay development, identification of novel substrates (e.g., phenoxytoluene, phenylallyether, and coumarone), and discovery of P450 BM-3 F87A mutants with 8-fold improvement in 3-phenoxytoluene hydroxylation activity. This activity increase was achieved by screening a saturation mutagenesis library of amino acid position Y51 using the 4-AAP protocol in the 96-well format.