A molecular dynamics simulation study of glycine/serine octapeptides labeled with 2,3-diazabicyclo[2.2.2]oct-2-ene fluorophore.

The compound 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) is a versatile fluorophore widely used in Förster resonance energy transfer (FRET) spectroscopy studies due to its remarkable sensitivity, enabling precise donor-acceptor distance measurements, even for short peptides. Integrating time-resolved and FRET spectroscopies with molecular dynamics simulations provides a robust approach to unravel the structure and dynamics of biopolymers in a solution. This study investigates the structural behavior of three octapeptide variants: Trp-(Gly-Ser)3-Dbo, Trp-(GlyGly)3-Dbo, and Trp-(SerSer)3-Dbo, where Dbo represents the DBO-containing modified aspartic acid, using molecular dynamics simulations. Glycine- and serine-rich amino acid fragments, common in flexible protein regions, play essential roles in functional properties. Results show excellent agreement between end-to-end distances, orientational factors from simulations, and the available experimental and theoretical data, validating the reliability of the GROMOS force field model. The end-to-end distribution, modeled using three Gaussian distributions, reveals a complex shape, confirmed by cluster analysis highlighting a limited number of significant conformations dominating the peptide landscape. All peptides predominantly adopt a disordered state in the solvent, yet exhibit a compact shape, aligning with the model of disordered polypeptide chains in poor solvents. Conformations show marginal dependence on chain composition, with Ser-only chains exhibiting slightly more elongation. This study enhances our understanding of peptide behavior, providing valuable insights into their structural dynamics in solution.

Modelling the adsorption of proteins to nanoparticles at the solid-liquid interface.

HYPOTHESIS: We developed a geometrical model to determine the theoretical maximum number of proteins that can pack as a monolayer surrounding a spherical nanoparticle. We applied our new model to study the adsorption of receptor binding domain (RBD) of the SARS-CoV-2 spike protein to silica nanoparticles. Due to its abundance and extensive use in manufacturing, silica represents a reservoir where the virus can accumulate. It is therefore important to study the adsorption and the persistence of viral components on inanimate surfaces. EXPERIMENTS: We used previously published datasets of nanoparticle-adsorbed proteins to validate the new model. We then used integrated experimental methods and Molecular Dynamics (MD) simulations to characterise binding of the RBD to silica nanoparticles and the effect of such binding on RBD structure. FINDINGS: The new model showed excellent fit with existing datasets and, combined to new RBD-silica nanoparticles binding data, revealed a surface occupancy of 32% with respect to the maximum RBD packing theoretically achievable. Up to 25% of RBD's secondary structures undergo conformational changes as a consequence of adsorption onto silica nanoparticles. Our findings will help developing a better understanding of the principles governing interaction of proteins with surfaces and can contribute to control the spread of SARS-CoV-2 through contaminated objects.

The Molecular Dynamics Simulation of Peptides on Gold Nanosurfaces.

This chapter contributes a short tutorial on the preparation of molecular dynamics (MD) simulations for a peptide in solution at the interface of an uncoated gold nanosurface. Specifically, the step-by-step procedure will give guidance to set up the simulation of a 16 amino acid long antimicrobial peptide on a gold layer using the program Gromacs for MD simulations.

Free Energy Profile of Domain Movement in Ligand-Free Citrate Synthase.

Citrate synthase plays a fundamental role in the metabolic cycle of the cell. Its catalytic mechanism is complex involving the binding of two substrates that cause a domain movement. In this paper, we used classical molecular dynamics simulations and umbrella-sampling simulations to determine the potential of mean force along a reaction coordinate for the domain movement in ligand-free citrate synthase from pig ( Sus scrofa). The results show that, at 293 K, the closed-domain conformation has a ∼4 kb T higher energy than the open-domain conformation. In a simple two-state model, this difference means that the enzyme spends 98% of the time in the open-domain conformation ready to receive the substrate, oxaloacetate, rather than the closed-domain conformation where the binding site would be inaccessible to the substrate. Given that experimental evidence indicates that the binding of oxaloacetate induces at least partial closure, this would imply an induced-fit mechanism which we argue is applicable to all enzymes with a functional domain movement for reasons of catalytic efficiency. A barrier of 4 kb T gives an estimation of the mean first passage time in the range 1-10 µs.

Modular assembly of proteins on nanoparticles.

Generally, the high diversity of protein properties necessitates the development of unique nanoparticle bio-conjugation methods, optimized for each different protein. Here we describe a universal bio-conjugation approach which makes use of a new recombinant fusion protein combining two distinct domains. The N-terminal part is Glutathione S-Transferase (GST) from Schistosoma japonicum, for which we identify and characterize the remarkable ability to bind gold nanoparticles (GNPs) by forming gold-sulfur bonds (Au-S). The C-terminal part of this multi-domain construct is the SpyCatcher from Streptococcus pyogenes, which provides the ability to capture recombinant proteins encoding a SpyTag. Here we show that SpyCatcher can be immobilized covalently on GNPs through GST without the loss of its full functionality. We then show that GST-SpyCatcher activated particles are able to covalently bind a SpyTag modified protein by simple mixing, through the spontaneous formation of an unusual isopeptide bond.

Adsorption mechanism of an antimicrobial peptide on carbonaceous surfaces: A molecular dynamics study.

Peptides are versatile molecules with applications spanning from biotechnology to nanomedicine. They exhibit a good capability to unbundle carbon nanotubes (CNT) by improving their solubility in water. Furthermore, they are a powerful drug delivery system since they can easily be uptaken by living cells, and their high surface-to-volume ratio facilitates the adsorption of molecules of different natures. Therefore, understanding the interaction mechanism between peptides and CNT is important for designing novel therapeutical agents. In this paper, the mechanisms of the adsorption of antimicrobial peptide Cecropin A-Magainin 2 (CA-MA) on a graphene nanosheet (GNS) and on an ultra-short single-walled CNT are characterized using molecular dynamics simulations. The results show that the peptide coats both GNS and CNT surfaces through preferential contacts with aromatic side chains. The peptide packs compactly on the carbon surfaces where the polar and functionalizable Lys side chains protrude into the bulk solvent. It is shown that the adsorption is strongly correlated to the loss of the peptide helical structure. In the case of the CNT, the outer surface is significantly more accessible for adsorption. Nevertheless when the outer surface is already covered by other peptides, a spontaneous diffusion, via the amidated C-terminus into the interior of the CNT, was observed within 150 ns of simulation time. We found that this spontaneous insertion into the CNT interior can be controlled by the polarity of the entrance rim. For the positively charged CA-MA peptide studied, hydrogenated and fluorinated rims, respectively, hinder and promote the insertion.

Molecular Properties of Astaxanthin in Water/Ethanol Solutions from Computer Simulations.

Astaxanthin (AXT) is a reference model of xanthophyll carotenoids, which is used in medicine and food industry, and has potential applications in nanotechnology. Because of its importance, there is a great interest in understanding its molecular properties and aggregation mechanism in water and mixed solvents. In this paper, we report a novel model of AXT for molecular dynamics simulation. The model is used to estimate different properties of the molecule in pure solutions and in water/ethanol mixtures. The calculated diffusion coefficients of AXT in pure water and ethanol are (3.22 ± 0.01) × 10(-6) cm(2) s(-1) and (2.7 ± 0.4) × 10(-6) cm(2) s(-1), respectively. Our simulations also show that the content of water plays a clear effect on the morphology of the AXT aggregation in water/ethanol mixture. In up to 75% (v/v) water concentration, a loosely connected network of dimers and trimers and two-dimensional array structures are observed. At higher water concentrations, AXT molecules form more compact three-dimensional structures that are preferentially solvated by the ethanol molecules. The ethanol preferential binding and the formation of a well connected hydrogen bonding network on these AXT clusters, suggest that such preferential solvation can play an important role in controlling the aggregate structure.

Strings-to-Rings Transition and Antiparallel Dipole Alignment in Two-Dimensional Methanols.

Structural order emerging in the liquid state necessitates a critical degree of anisotropy of the molecules. For example, liquid crystals and Langmuir monolayers require rod- or disc-shaped and long-chain amphiphilic molecules, respectively, to break the isotropic symmetry of liquids. In this Letter we present results from molecular dynamics simulations demonstrating that in two-dimensional liquids, a significantly smaller degree of anisotropy is sufficient to allow structural organization. In fact, the condensed phase of the smallest amphiphilic molecule, methanol, confined between two, or adsorbed on, graphene sheets forms a monolayer characterized by long chains of molecules. Intrachain interactions are dominated by hydrogen bonds, whereas interchain interactions are dispersive. Upon a decrease in density toward a gaslike state, these strings are transformed into rings. The two-dimensional liquid phase of methanol undergoes another transition upon cooling; in this case, the order-disorder transition is characterized by a low-temperature phase in which the hydrogen bond dipoles of neighboring strings adopt an antiparallel orientation.

Iterative key-residues interrogation of a phytase with thermostability increasing substitutions identified in directed evolution.

Bacterial phytases have attracted industrial interest as animal feed supplement due to their high activity and sufficient thermostability (required for feed pelleting). We devised an approach named KeySIDE,  an iterative Key-residues interrogation of the wild type with Substitutions Identified in Directed Evolution for improving Yersinia mollaretii phytase (Ymphytase) thermostability by combining key beneficial substitutions and elucidating their individual roles. Directed evolution yielded in a discovery of nine positions in Ymphytase and combined iteratively to identify key positions. The "best" combination (M6: T77K, Q154H, G187S, and K289Q) resulted in significantly improved thermal resistance; the residual activity improved from 35 % (wild type) to 89 % (M6) at 58 °C and 20-min incubation. Melting temperature increased by 3 °C in M6 without a loss of specific activity. Molecular dynamics simulation studies revealed reduced flexibility in the loops located next to helices (B, F, and K) which possess substitutions (Helix-B: T77K, Helix-F: G187S, and Helix-K: K289E/Q). Reduced flexibility in the loops might be caused by strengthened hydrogen bonding network (e.g., G187S and K289E/K289Q) and a salt bridge (T77K). Our results demonstrate a promising approach to design phytases in food research, and we hope that the KeySIDE might become an attractive approach for understanding of structure-function relationships of enzymes.

Unraveling Binding Effects of Cobalt(II) Sepulchrate with the Monooxygenase P450 BM-3 Heme Domain Using Molecular Dynamics Simulations.

One of the major limitations to exploit enzymes in industrial processes is their dependence on expensive reduction equivalents like NADPH to drive their catalytic cycle. Soluble electron-transfer (ET) mediators like cobalt(II) sepulchrate have been proposed as a cost-effective alternative to shuttle electrons between an inexpensive electron source and an enzyme's redox center. The interactions of these molecules with enzymes have not yet been elucidated at the molecular level. Herein, molecular dynamics simulations are performed to understand the binding and ET mechanism of the cobalt(II) sepulchrate with the heme domain of cytochrome P450 BM-3. The study provides a detailed map of ET mediator binding sites on the protein surface that are prevalently composed of Asp and Glu amino acids. The cobalt(II) sepulchrate does not show a preferential binding to these sites. However, among the observed binding sites, only few of them provide efficient ET pathways to heme iron. The results of this study can be used to improve the ET mediator efficiency of the enzyme for possible biotechnological applications.

Structure, dynamics, and function of the monooxygenase P450 BM-3: insights from computer simulations studies.

The monooxygenase P450 BM-3 is a NADPH-dependent fatty acid hydroxylase enzyme isolated from soil bacterium Bacillus megaterium. As a pivotal member of cytochrome P450 superfamily, it has been intensely studied for the comprehension of structure-dynamics-function relationships in this class of enzymes. In addition, due to its peculiar properties, it is also a promising enzyme for biochemical and biomedical applications. However, despite the efforts, the full understanding of the enzyme structure and dynamics is not yet achieved. Computational studies, particularly molecular dynamics (MD) simulations, have importantly contributed to this endeavor by providing new insights at an atomic level regarding the correlations between structure, dynamics, and function of the protein. This topical review summarizes computational studies based on MD simulations of the cytochrome P450 BM-3 and gives an outlook on future directions.

Cosolvent, ions, and temperature effects on the structural properties of Cecropin A-Magainin 2 hybrid peptide in solutions.

Antimicrobial peptides are promising alternative to traditional antibiotics and antitumor drugs for the battle against new antibiotic resistant bacteria strains and cancer maladies. The study of their structural and dynamics properties at physiological conditions can help to understand their stability, delivery mechanisms, and activity in the human body. In this article, we have used molecular dynamics simulations to study the effects of solvent environment, temperature, ions concentration, and peptide concentration on the structural properties of the antimicrobial hybrid peptide Cecropin A-Magainin 2. In TFE/water mixtures, the structure of the peptide retained α-helix contents and an average hinge angle in close agreement with the experimental NMR and CD measurements reported in literature. Compared to the TFE/water mixture, the peptide simulated at the same ionic concentration lost most of its α-helix structure. The increase of peptide concentration at both 300 and 310 K resulted in the peptide aggregation. The peptides in the complex retained the initial N-ter α-helix segment during all the simulation. The α-helix stabilization is further enhanced in the high salt concentration simulations. The peptide aggregation was not observed in TFE/water mixture simulations and, the peptide aggregate, obtained from the water simulation, simulated in the same conditions did dissolve within few tens of nanoseconds. The results of this study provide insights at molecular level on the structural and dynamics properties of the CA-MA peptide at physiological and membrane mimic conditions that can help to better understand its delivery and interaction with biological interfaces.

The Mutagenesis Assistant Program.

Mutagenesis Assistant Program (MAP) is a web-based statistical tool to develop directed evolution strategies by investigating the consequences at the amino acid level of the mutational biases of random mutagenesis methods on any given gene. The latest development of the program, the MAP(2.0)3D server, correlates the generated amino acid substitution patterns of a specific random mutagenesis method to the sequence and structural information of the target protein. The combined information can be used to select an experimental strategy that improves the chances of obtaining functionally efficient and/or stable enzyme variants. Hence, the MAP(2.0)3D server facilitates the "in silico" prescreening of the target gene by predicting the amino acid diversity generated in a random mutagenesis library. Here, we describe the features of MAP(2.0)3D server by analyzing, as an example, the cytochrome P450BM3 monooxygenase (CYP102A1). The MAP(2.0)3D server is available publicly at http://map.jacobs-university.de/map3d.html.

Micellar drug nanocarriers and biomembranes: how do they interact?

Pluronic based formulations are among the most successful nanomedicines and block-copolymer micelles including drugs that are undergoing phase I/II studies as anticancer agents. Using coarse-grained models, molecular dynamics simulations of large-scale systems, modeling Pluronic micelles interacting with DPPC lipid bilayers, on the µs timescale have been performed. Simulations show, in agreement with experiments, the release of Pluronic chains from the micelle to the bilayer. This release changes the size of the micelle. Moreover, the presence of drug molecules inside the core of the micelle has a strong influence on this process. The picture emerging from the simulations is that the micelle stability is a result of an interplay of drug-micelle core and block-copolymer-bilayer interactions. The equilibrium size of the drug vector shows a strong dependency on the hydrophobicity of the drug molecules embedded in the core of the micelle. In particular, the radius of the micelle shows an abrupt increase in a very narrow range of drug molecule hydrophobicity.

Insight into the redox partner interaction mechanism in cytochrome P450BM-3 using molecular dynamics simulations.

Flavocytochrome P450BM-3 is a soluble bacterial reductase composed of two flavin (FAD/FMN) and one HEME domains. In this article, we have performed molecular dynamics simulations on both the isolated FMN and HEME domains and their crystallographic complex, with the aim to study their binding modes and to garner insight into the interdomain electron transfer (ET) mechanism. The results evidenced an interdomain conformational rearrangement that reduces the average distance between the FMN and HEME cofactors from 1.81 nm, in the crystal structure, to an average value of 1.41±0.09 nm along the simulation. This modification is in agreement with previously proposed hypotheses suggesting that the crystallographic FMN/HEME complex is not in the optimal arrangement for favorable ET rate under physiological conditions. The calculation of the transfer rate along the simulation, using the Pathways Path method, demonstrated the occurrence of seven ET pathways between the two redox centers, with three of them providing ET rates (KET ) comparable with the experimental one. The sampled ET pathways comprise the amino acids N319, L322, F390, K391, P392, F393, A399, C400, and Q403 of the HEME domain and M490 of the FMN domain. The values of KET closer to the experiment were found along the pathways FMN(C7)→F390→K391→P392→HEME(Fe) and FMN(C8)→M490→F393→HEME(Fe). Finally, the analysis of the collective modes of the protein complex evidences a clear correlation of the first two essential modes with the activation of the most effective ET pathways along the trajectory.

Insights on activity and stability of subtilisin E towards guanidinium chloride and sodium dodecylsulfate.

A subtilisin E variant (M4) showing high activity and resistance towards guanidinium chloride (GdmCl) and sodium dodecylsulfate (SDS) was previously identified after three rounds of directed evolution [Li et al., ChemBioChem 2012, 13(5), 691-699.]. In this report, 10 additional positions, identified during directed subtilisin E evolution, were saturated on the previously reported SeSaM1-5 variant (S62/A153/G166/I205). Screening confirmed that chaotolerant variants included amino acid substitutions either in the active site, or the substrate binding pocket. Two variants, M5 (S62I/A153V/G166S/T224A/T240S) and M6 (S62I/A153V/G166S/I205V/N218S/T224A) were finally generated to maximize activity and stability in the presence of GdmCl or SDS. The inactivation concentration (IC50) of M6 using Suc-AAPF-pNA as substrate was significantly increased compared to M4 in the presence of GdmCl (IC50 (M4): 2.7M; IC50 (M6): 4.6M) and SDS (IC50 (M4): 1.5%; IC50 (M6): 4.0%). The half-life in 5M GdmCl was also significantly improved for M6 compared to M4 (t 1/2 (M4): 2min; t 1/2 (M6): 15min). M5 retained resistance towards GdmCl or SDS as in M4. The activity of M5 towards a complex protein substrate (Azocasein) was increased by ∼1.5 fold compared to M4 and M6. Circular dichroism (CD) analysis for subtilisin E wild type (WT) and three variants (M4, M5 and M6) indicated that secondary structures of all variants including wild type at 1-2M GdmCl (except M4) were not significantly perturbed, with unfolding occurring for WT and all three variants above 3M GdmCl. In SDS, the secondary structures of WT and all three variants remained intact at concentrations of 0.5 to 2.0% (w/v) SDS. Results suggest that subtilisin E inactivation occurred most likely due to inhibitory effect, since a general unfolding of the enzyme was not observed through circular dichroism. Such inhibition could be avoided by limiting the access of GdmCl and SDS to the active site and/or to residues involved in substrate binding.

Theoretical study of binding and permeation of ether-based polymers through interfaces.

We present a molecular dynamics simulation study on the interactions of poly(ethylene oxide) (PEO), poly(propylene oxide) (PPO), and their ABA-type block copolymer, poloxamers, at water/n-heptane and 1,2-dimyristoyl-sn-glycero-3-phospatidycholine (DMPC) lipid bilayer/water interfaces. The partition coefficients in water/1-octanol of the linear polyethers up to three monomers were calculated. The partition coefficients evidenced a higher hydrophobicity of the PPO in comparison to PEO. At the water/n-heptane interface, the polymers tend to adopt elongated conformations in agreement with similar experimental ellipsometry studies of different poloxamers. In the case of the poloxamers at the n-heptane/water interface, the stronger preference of the PPO block for the hydrophobic phase resulted in bottle-brush-type polymer conformations. At lipid bilayer/water interface, the PEO polymers, as expected from their hydrophilic nature, are weakly adsorbed on the surface of the lipid bilayer and locate in the water phase close to the headgroups. The free energy barriers of permeation calculated for short polymer chains suggest a thermodynamics propensity for the water phase that increase with the chain length. The lower affinity of PEO for the hydrophobic interior of the lipid bilayer resulted in the spontaneous expulsion within the simulation time. On the contrary, PPO chains and poloxamers have a longer residence time inside the bilayer, and they tend to concentrate in the tail region of the bilayer near the polar headgroups. In addition, polymers with PPO unit length comparable to the thickness of the hydrophobic region of the bilayer tend to span across the bilayer.

P450 BM3 crystal structures reveal the role of the charged surface residue Lys/Arg184 in inversion of enantioselective styrene epoxidation.

Solved crystal structures of P450 BM3 variants in complex with styrene provide on the molecular level a first explanation of how a positively charged surface residue inverts the enantiopreference of styrene epoxidation. The obtained insights into productive and non-productive styrene binding modes deepened our understanding of enantioselective epoxidation with P450 BM3.

Interaction of Curcumin with PEO-PPO-PEO block copolymers: a molecular dynamics study.

Curcumin, a naturally occurring drug molecule, has been extensively investigated for its various potential usages in medicine. Its water insolubility and high metabolism rate require the use of drug delivery systems to make it effective in the human body. Among various types of nanocarriers, block copolymer based ones are the most effective. These polymers are broadly used as drug-delivery systems, but the nature of this process is poorly understood. In this paper, we propose a molecular dynamics simulation study of the interaction of Curcumin with block copolymer based on polyethylene oxide (PEO) and polypropylene oxide (PPO). The study has been conducted considering the smallest PEO and PPO oligomers and multiple chains of the block copolymer Pluronic P85. Our study shows that the more hydrophobic 1,2-dimethoxypropane (DMP) molecules and PPO block preferentially coat the Curcumin molecule. In the case of the Pluronic P85, simulation shows formation of a drug-polymer aggregate within 50 ns. This process leaves exposed the PEO part of the polymers, resulting in better solvation and stability of the drug in water.

Conformational Dynamics of the FMN-Binding Reductase Domain of Monooxygenase P450BM-3.

In the cytochrome P450BM-3, the flavin mononucleotide (FMN) binding domain is an intermediate electron donor between the flavin adenine dinucleotide (FAD) binding domain and the HEME domain. Experimental evidence has shown that different redox states of FMN cofactor were found to induce conformational changes in the FMN domain. Herein, molecular dynamics (MD) simulation is used to gain insight into the latter phenomenon at the atomistic level. We have studied the effect of FMN cofactor and its redox states (oxidized and reduced) on the structure and dynamics of the FMN domain. The results of our study show significant differences in the atomic fluctuation amplitude of the FMN domain in both holo- and apoprotein. The change in the protonation state of FMN cofactor mostly affects its binding in holo-protein. In particular, the loops involved in the binding of the isoalloxazine ring (Lß4) and ribityl side chain (Lß1) adopt different conformations in both reduced and oxidized states. In addition, the reduced FMN cofactor mainly induces a conformational change in Trp574 residue (Lß4) that is essential for controlling electron transfer (ET) within P450BM-3 domains. The structure of the apoprotein in solution remains mostly unchanged with respect to the crystal structure of the holo-protein. However, FMN binding loops were more flexible in apoprotein that might favor the rebinding of FMN cofactor. In the holo-protein simulation, the largest conformational changes in FMN cofactor are caused by the ribityl side chain. The isoalloxazine ring of FMN cofactor remains almost planar (∼177°) in the oxidized state and bends along the N5-N10 axis at an angle of ∼160° in the reduced state. The collective modes of the isoalloxazine ring were identical in both protonation states of FMN cofactor except the first eigenvector. In the reduced state, the isoalloxazine ring attains the butterfly motion as a dominant collective motion in the first eigenvector due to the bending along the N5-N10 axis.