Biomedical subjects

Danilo Roccatano

Publications and source records attributed to Danilo Roccatano.

At least 19 recordsLinked to original sources

PubMed2007

Are transversion mutations better? A Mutagenesis Assistant Program analysis on P450 BM-3 heme domain.

Directed evolution represents a versatile tool to tailor enzyme properties to needs in industrial applications and to understand structure-function relationships. Genetic diversity is commonly generated using error-prone PCR. Exploration of sequence space by random mutagenesis strongly favors transitions when enzyme-based mutagenesis methods are employed (Wong, T. S., Zhurina, D., Schwaneberg, U., Comb. Chem. High Throughput Screen. 2006, 9, 271-288). The genetic code has been organized in a manner that limits chemical diversity when a single transition mutation occurs in a codon (Wong, T. S., Roccatano, D., Schwaneberg, U., Biocatal. Biotransformation 2006, in press). Are transitions more beneficial than transversions for adapting biocatalysts to non-natural process conditions? In a statistical analysis performed with the Mutagenesis Assistant Program (MAP), we compared the consequences of transition and transversion bias on amino acid substitution patterns of the P450 BM-3 heme domain. For the analysis, we used a recently introduced benchmarking system consisting of a protein structure indicator, an amino acid diversity indicator with a codon diversity coefficient, and a chemical diversity indicator. A detailed analysis for the P450 BM-3 heme domain showed that an ideal transversion bias generates more diverse amino acid substitution patterns with a significantly different chemical composition than an ideal transition bias. Emphasis is given on the theoretical analysis with a brief discussion on potential implication of transition and transversion bias in directed evolution experiments.

Algorithms↗

PubMed2006

Synthetic polymers and biomembranes. How do they interact? Atomistic molecular dynamics simulation study of PEO in contact with a DMPC lipid bilayer.

The understanding of interactions of poly(ethylene glycol) (PEG) or poly(ethylene oxide) (PEO) with biological interfaces has important technological application in industry and in medicine. In this paper, structural and dynamical properties of PEO at the dimyristoylphospatidylcholine (DMPC) bilayer/water interface have been investigated by molecular dynamics (MD) and steered molecular dynamics (SMD) simulations. The structural properties of a PEO chain in bulk water, at the water/vacuum interface, and in the presence of the membrane were compared with available experimental data. The presence of a barrier for the PEO penetration into the DMPC bilayer has been found. A qualitative estimation of the barrier provided a value equal to approximately 19 kJ/mol, that is, 7 times the value of kT at 310 K.

Dimyristoylphosphatidylcholine↗

PubMed2006

Toward understanding the inactivation mechanism of monooxygenase P450 BM-3 by organic cosolvents: a molecular dynamics simulation study.

Cytochrome P450 BM-3 from Bacillus megaterium is an extensively studied enzyme for industrial applications. A major focus of current protein engineering research is directed to improving the catalytic performance of P450 BM-3 toward nonnatural substrates of industrial importance in the presence of organic solvents or cosolvents. For the latter reason, it is important to study the effect of organic cosolvent molecules on the structure and dynamics of the enzyme, in particular, the effect of cosolvent molecules on the active site's structure and dynamics. In this paper, we have studied, using molecular dynamics (MD) simulations, the F87A mutant of P450 BM-3 in the presence of DMSO as cosolvent, to understand the role of the F87A substitution for its catalytic activity. This mutant exhibits an altered regioselectivity and substrate specificity compared with wild-type; however, it has lower tolerance toward DMSO. The simulation results offer an explanation for the DMSO sensitivity of the F87A mutant. Our simulation results show that the F87 side chain prevents the disturbance of the water molecule bound to the heme iron by DMSO molecules. The absence of the phenyl ring in F87A mutant promotes interactions of the DMSO molecule with the heme iron resulting in water displacement by DMSO at the catalytic heme center.

Bacillus megaterium↗

PubMed2006

Distance distributions of short polypeptides recovered by fluorescence resonance energy transfer in the 10 A domain.

Fluorescence resonance energy transfer (FRET) between tryptophan (Trp) as donor and 2,3-diazabicyclo[2.2.2]oct-2-ene (Dbo) as acceptor was studied by steady-state and time-resolved fluorescence spectroscopy. The unique feature of this FRET pair is its exceptionally short Förster radius (10 A), which allows one to recover distance distributions in very short structureless peptides. The technique was applied to Trp-(GlySer)n-Dbo-NH2 peptides with n = 0-10, for which the average probe/quencher distance ranged between 8.7 and 13.7 A experimentally (in propylene glycol, analysis according to wormlike chain model) and 8.6-10.2 A theoretically (for n = 0-6, GROMOS96 molecular dynamics simulations). The larger FRET efficiency in steady-state compared to time-resolved fluorescence experiments was attributed to a static quenching component, suggesting that a small but significant part (ca. 10%) of the conformations are already in van der Waals contact when excitation occurs.

Bridged Bicyclo Compounds, Heterocyclic↗

PubMed2005

A statistical analysis of random mutagenesis methods used for directed protein evolution.

We have developed a statistical method named MAP (mutagenesis assistant program) to equip protein engineers with a tool to develop promising directed evolution strategies by comparing 19 mutagenesis methods. Instead of conventional transition/transversion bias indicators as benchmarks for comparison, we propose to use three indicators based on the subset of amino acid substitutions generated on the protein level: (1) protein structure indicator; (2) amino acid diversity indicator with a codon diversity coefficient; and (3) chemical diversity indicator. A MAP analysis for a single nucleotide substitution was performed for four genes: (1) heme domain of cytochrome P450 BM-3 from Bacillus megaterium (EC 1.14.14.1); (2) glucose oxidase from Aspergillus niger (EC 1.1.3.4); (3) arylesterase from Pseudomonas fluorescens (EC 3.1.1.2); and (4) alcohol dehydrogenase from Saccharomyces cerevisiae (EC 1.1.1.1). Based on the MAP analysis of these four genes, 19 mutagenesis methods have been evaluated and criteria for an ideal mutagenesis method have been proposed. The statistical analysis showed that existing gene mutagenesis methods are limited and highly biased. An average amino acid substitution per residue of only 3.15-7.4 can be achieved with current random mutagenesis methods. For the four investigated gene sequences, an average fraction of amino acid substitutions of 0.5-7% results in stop codons and 4.5-23.9% in glycine or proline residues. An average fraction of 16.2-44.2% of the amino acid substitutions are preserved, and 45.6% (epPCR method) are chemically different. The diversity remains low even when applying a non-biased method: an average of seven amino acid substitutions per residue, 2.9-4.7% stop codons, 11.1-16% glycine/proline residues, 21-25.8% preserved amino acids, and 55.5% are amino acids with chemically different side-chains. Statistical information for each mutagenesis method can further be used to investigate the mutational spectra in protein regions regarded as important for the property of interest.

Amino Acid Substitution↗

PubMed2005

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

The molecular mechanism by which HFIP stabilizes the alpha-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 alpha-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.

Computer Simulation↗

PubMed2005

Molecular dynamics simulation of water near nanostructured hydrophobic surfaces: interfacial energies.

We present results from molecular dynamics simulations of water near structured hydrophobic surfaces. The surface structures reported herein are a planar alkane crystal as a reference and crystals with a hole and a protrusion of approximately 2.5 nm diameter and 0.5 nm depth or height. All indicators show that surface structuring increases the hydrophobicity: The water density is reduced near the structure elements, and the number of residual contacts between water and the surface decreases by about 40 % with respect to the planar surface. Thermodynamic integration shows that the interfacial energy of the structured surfaces is about 7 mJ m(-2) higher for structured surfaces than for the planar surface. The hydrophobicity increases by a similar amount for the hole and the protrusion geometries compared to the planar surface.

Alkanes↗

PubMed2005

Structural and dynamic properties of cytochrome P450 BM-3 in pure water and in a dimethylsulfoxide/water mixture.

Solvent molecules play an important role for the structural and dynamical properties of proteins. A major focus of current protein engineering is the development of enzymes that are catalytically active in the presence of organic solvents. The monooxygenase P450 BM-3 is one of the best-studied enzymes and promising for industrial applications but with limited activity in the presence of organic solvents or cosolvents. To gain insights into the structural and dynamical properties of the heme domain of this enzyme in solution, molecular dynamics simulations in pure water and in a 14% DMSO/water mixture were performed. The results of the simulations show overall similar structural fluctuations in both solvent systems, with no indication of partial or global unfolding. In 14% DMSO, the regions comprising the helices E, F, and the EF loop (implicated in controlling the entry to the active site channel) undergo a large shift. Significant changes were also observed near the active site access channel at the residue R47. During the simulation, no DMSO molecule penetrated the active site. However, a significant accumulation of DMSO molecules close to the substrate-binding site and to the Flavin Mononucleotide (FMN) reductase domain interface was observed.

Bacillus megaterium↗

PubMed2005

Conformational and electronic properties of a microperoxidase in aqueous solution: a computational study.

A theoretical study of the conformational properties of a small heme peptide in aqueous solution is carried out by classical, long-timescale molecular dynamics simulations. The electronic properties of this species, that is, the relative energies of its excited electronic states and the redox potential, are reproduced and related to the conformational behavior using the perturbed matrix method and basic statistical mechanics. Our results show an interesting coupling between the conformational transitions and the electronic properties. These investigations, beyond the biophysically relevant results addressing the long-standing question of the actual role of the enzyme structure on the enzyme activity, are also of some methodological interest since they offer a further computational perspective for including the electronic degrees of freedom into the modeling of rather complex molecular systems.

Biophysics↗

PubMed2005

Dynamical aspects of TEM-1 beta-lactamase probed by molecular dynamics.

The dynamical aspects of the fully hydrated TEM-1 beta-lactamase have been determined by a 5 ns Molecular Dynamics simulation. Starting from the crystallographic coordinates, the protein shows a relaxation in water with an overall root mean square deviation from the crystal structure increasing up to 0.17 nm, within the first nanosecond. Then a plateau is reached and the molecule fluctuates around an equilibrium conformation. The results obtained in the first nanosecond are in agreement with those of a previous simulation (Diaz et al., J. Am. Chem. Soc., (2003) 125, 672-684). The successive equilibrium conformation in solution shows an increased mobility characterized by the following aspects. A flap-like translational motion anchors the omega-loop to the body of the enzyme. A relevant part of the backbone dynamics implies a rotational motion of one domain relative to the other. The water molecules in the active site can exchange with different residence times. The H-bonding networks formed by the catalytic residues are frequently interrupted by water molecules that could favour proton transfer reactions. An additional simulation, where the aspartyl dyad D214-D233 was considered fully deprotonated, shows that the active site is destabilized.

Catalytic Domain↗

PubMed2004

Beta-hairpin conformation of fibrillogenic peptides: structure and alpha-beta 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 alpha-helical to beta-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 Abeta(12-28) fragment from the Abeta(1-42) peptide responsible for Alzheimer's disease. The simulations highlight the unfolding of alpha-helices, followed by the formation of bent conformations and a final convergence to ordered in register beta-hairpin conformations. The beta-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 beta-hairpin structures. Furthermore the simulations presented herein evidence the stabilization of the alpha-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.

Animals↗

PubMed2004

Investigating the accessibility of the closed domain conformation of citrate synthase using essential dynamics sampling.

A molecular dynamics study of pig heart citrate synthase is presented that aims to directly address the question of whether, for this enzyme, the ligand-induced closed domain conformation is accessible to the open unliganded enzyme. The approach utilises the technique of essential dynamics sampling, which is used in two modes. In exploring mode, the enzyme is encouraged to explore domain conformations it might not normally sample in free molecular dynamics simulation. In targeting mode, the enzyme is encouraged to adopt the domain conformation of a target structure. Using both modes extensively, it has been found that when the enzyme is prepared from a crystallographic open-domain structure and is in the unliganded state, it is unable to adopt the crystallographic closed-domain conformation of the liganded enzyme. Likewise, when the enzyme is prepared from the crystallographic closed liganded conformation with the ligands removed, it is unable to adopt the crystallographic open domain conformation. Structural investigations point to a common structural difference that is the source of this energy barrier; namely, the shift of alpha-helix 328-341 along its own axis relative to the large domain. Without this shift, the domains are unable to close or open fully. The charged substrate, oxaloacetate, binds near the base of this helix in the large domain and the interaction of Arg329 at the base of the helix with oxaloacetate is one that is consistent with the shift of this helix in going from the crystallographic open to closed structure. Therefore, the results suggest that without the substrate the enzyme remains in a partially open conformation ready to receive the substrate. In this way, the efficiency of the enzyme should be increased over one that is closed part of the time, with its binding site inaccessible to the substrate.

Citrate (si)-Synthase↗

PubMed2004

Molecular dynamics simulation of the interaction between the complex iron-sulfur flavoprotein glutamate synthase and its substrates.

Glutamate synthase (GltS) is a complex iron-sulfur flavoprotein that catalyzes the reductive transfer of L-glutamine amide group to the C2 carbon of 2-oxoglutarate yielding two molecules of L-glutamate. Molecular dynamics calculations in explicit solvent were carried out to gain insight into the conformational flexibility of GltS and into the role played by the enzyme substrates in regulating the catalytic cycle. We have modelled the free (unliganded) form of Azospirillum brasilense GltS alpha subunit and the structure of the reduced enzyme in complex with the L-glutamine and 2-oxoglutarate substrates starting from the crystallographically determined coordinates of the GltS alpha subunit in complex with L-methionine sulphone and 2-oxoglutarate. The present 4-ns molecular dynamics calculations reveal that the GltS glutaminase site may exist in a catalytically inactive conformation unable to bind glutamine, and in a catalytically competent conformation, which is stabilized by the glutamine substrate. Substrates binding also induce (1) closure of the loop formed by residues 263-271 with partial shielding of the glutaminase site from solvent, and (2) widening of the ammonia tunnel entrance at the glutaminase end to allow for ammonia diffusion toward the synthase site. The Q-loop of glutamate synthase, which acts as an active site lid in other amidotransferases, seems to maintain an open conformation. Finally, binding of L-methionine sulfone, a glutamine analog that mimics the tetrahedral transient species occurring during its hydrolysis, causes a coordinated rigid-body motion of segments of the glutaminase domain that results in the inactive conformation observed in the crystal structure of GltS alpha subunit.

Azospirillum brasilense↗

PubMed2004

Extended molecular dynamics simulation of the carbon monoxide migration in sperm whale myoglobin.

We report the results of an extended molecular dynamics simulation on the migration of photodissociated carbon monoxide in wild-type sperm whale myoglobin. Our results allow following one possible ligand migration dynamics from the distal pocket to the Xe1 cavity via a path involving the other xenon binding cavities and momentarily two additional packing defects along the pathway. Comparison with recent time resolved structural data obtained by Laue crystallography with subnanosecond to millisecond resolution shows a more than satisfactory agreement. In fact, according to time resolved crystallography, CO, after photolysis, can occupy the Xe1 and Xe4 cavities. However, no information on the trajectory of the ligand from the distal pocket to the Xe1 is available. Our results clearly show one possible path within the protein. In addition, although our data refer to a single trajectory, the local dynamics of the ligand in each cavity is sufficiently equilibrated to obtain local structural and thermodynamic information not accessible to crystallography. In particular, we show that the CO motion and the protein fluctuations are strictly correlated: free energy calculations of the migration between adjacent cavities show that the migration is not a simple diffusion but is kinetically or thermodynamically driven by the collective motions of the protein; conversely, the protein fluctuations are influenced by the ligand in such a way that the opening/closure of the passage between adjacent cavities is strictly correlated to the presence of CO in its proximity. The compatibility between time resolved crystallographic experiments and molecular dynamics simulations paves the way to a deeper understanding of the role of internal dynamics and packing defects in the control of ligand binding in heme proteins.

Animals↗

PubMed2003

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.

Algorithms↗

PubMed2003

Selective excitation of native fluctuations during thermal unfolding simulations: horse heart cytochrome c as a case study.

The effect of temperature on the activation of native fluctuation motions during molecular dynamics unfolding simulations of horse heart cytochrome c has been studied. Essential dynamics analysis has been used to analyze the preferred directions of motion along the unfolding trajectories obtained by high temperature simulations. The results of this study have evidenced a clear correlation between the directions of the deformation motions that occur in the first stage of the unfolding process and few specific essential motions characterizing the 300 K dynamics of the protein. In particular, one of those collective motions, involved in the fluctuation of a loop region, is specifically excited in the thermal denaturation process, becoming progressively dominant during the first 500 ps of the unfolding simulations. As further evidence, the essential dynamics sampling performed along this collective motion has shown a tendency of the protein to promptly unfold. According to these results, the mechanism of thermal induced denaturation process involves the selective excitation of one or few specific equilibrium collective motions.

Animals↗

PubMed2003

Molecular dynamics simulations of lignin peroxidase in solution.

The dynamical and structural properties of lignin peroxidase and its Trp171Ala mutant have been investigated in aqueous solution using molecular dynamics (MD) simulations. In both cases, the enzyme retained its overall backbone structure and all its noncovalent interactions in the course of the MD simulations. Very interestingly, the analysis of the MD trajectories showed the presence of large fluctuations in correspondence of the residues forming the heme access channel; these movements enlarge the opening and facilitate the access of substrates to the enzyme active site. Moreover, steered molecular dynamics docking simulations have shown that lignin peroxidase natural substrate (veratryl alcohol) can easily approach the heme edge through the access channel.

Benzyl Alcohols↗

PubMed2003

Interplay between hydrophobic cluster and loop propensity in beta-hairpin formation: a mechanistic study.

We investigated the structural determinants of the stability of a designed beta-hairpin containing a natural hydrophobic cluster from the protein GB1 and a D-Pro-Gly turn forming sequence. The results of our simulations shed light on the factors leading to an ordered secondary structure in a model peptide: in particular, the importance of the so-called diagonal interactions in forming a stable hydrophobic nucleus in the beta-hairpin, together with the more obvious lateral interactions, is examined. With the use of long timescale MD simulations in explicit water, we show the role of diagonal interactions in driving the peptide to the correct folded structure (formation of the hydrophobic core with Trp 2, Tyr 4, and Phe 9 in the first stages of refolding) and in keeping it in the ensemble of folded conformations. The combination of the stabilizing effects of the D-Pro-Gly turn sequence and of the hydrophobic nucleus formation thus favors the attainment of an ordered secondary structure compatible with the one determined experimentally. Moreover, our data underline the importance of the juxtapositions of the side chains of amino acids not directly facing each other in the three-dimensional structure. The combination of these interactions forces the peptide to sample a nonrandom portion of the conformational space, as can be seen in the rapid collapse to an ordered structure in the refolding simulation, and shows that the unfolded state can be closely correlated to the folded ensemble of structures, at least in the case of small model peptides.

Computer Simulation↗