Kinetics, statistics, and energetics of lipid membrane electroporation studied by molecular dynamics simulations

Membrane electroporation is the method to directly transfer bioactive substances such as drugs and genes into living cells, as well as preceding electrofusion. Although much information on the microscopic mechanism has been obtained both from experiment and simulation, the existence and nature of possible intermediates is still unclear. To elucidate intermediates of electropore formation by direct comparison with measured prepore formation kinetics, we have carried out 49 atomistic electroporation simulations on a palmitoyl-oleoyl-phosphatidylcholine bilayer for electric field strengths between 0.04 and 0.7 V/nm. A statistical theory is developed to facilitate direct comparison of experimental (macroscopic) prepore formation kinetics with the (single event) preporation times derived from the simulations, which also allows us to extract an effective number of lipids involved in each pore formation event. A linear dependency of the activation energy for prepore formation on the applied field is seen, with quantitative agreement between experiment and simulation. The distribution of preporation times suggests a four-state pore formation model. The model involves a first intermediate characterized by a differential tilt of the polar lipid headgroups on both leaflets, and a second intermediate (prepore), where a polar chain across the bilayer is formed by 3-4 lipid headgroups and several water molecules, thereby providing a microscopic explanation for the polarizable volume derived previously from the measured kinetics. An average pore radius of 0.47 ± 0.15 nm is seen, in favorable agreement with conductance measurements and electrooptical experiments of lipid vesicles.     

Comparative molecular dynamics of mesophilic and psychrophilic protein homologues studied by 1.2 ns simulations

It is well established that the dynamic motion of proteins plays an important functional role, and that the adaptation of a protein molecule to its environment requires optimization of internal non-covalent interactions and protein-solvent interactions. Serine proteinases in general, and trypsin in particular has been used as a model system in exploring possible structural features for cold adaptation. In this study, a 500 p.s. and a 1200 p.s. molecular dynamics (MD) simulation at 300 K of both anionic salmon trypsin and cationic bovine trypsin are analyzed in terms of molecular flexibility, internal non-covalent interactions and protein-solvent interactions. The present MD simulations do not indicate any increased flexibility of the cold adapted enzyme on an overall basis. However, the apparent higher flexibility and deformability of the active site of anionic salmon trypsin may lower the activation energy for ligand binding and for catalysis, and might be a reason for the increased binding affinity and catalytic efficiency compared to cationic bovine trypsin.     

Virus capsid dissolution studied by microsecond molecular dynamics simulations

Dissolution of many plant viruses is thought to start with swelling of the capsid caused by calcium removal following infection, but no high-resolution structures of swollen capsids exist. Here we have used microsecond all-atom molecular simulations to describe the dynamics of the capsid of satellite tobacco necrosis virus with and without the 92 structural calcium ions. The capsid expanded 2.5% upon removal of the calcium, in good agreement with experimental estimates. The water permeability of the native capsid was similar to that of a phospholipid membrane, but the permeability increased 10-fold after removing the calcium, predominantly between the 2-fold and 3-fold related subunits. The two calcium binding sites close to the icosahedral 3-fold symmetry axis were pivotal in the expansion and capsid-opening process, while the binding site on the 5-fold axis changed little structurally. These findings suggest that the dissociation of the capsid is initiated at the 3-fold axis.     

Ribonucleotide reductase inhibition by p-alkoxyphenols studied by molecular docking and molecular dynamics simulations

Ribonucleotide reductase (RNR) is necessary for production of the precursor deoxyribonucleotides for DNA synthesis. Class Ia RNR functions via a stable free radical in one of the two components protein R2. The enzyme mechanism involves long range (proton coupled) electron transfer between protein R1 and the tyrosyl radical in protein R2. Earlier experimental studies showed that p-alkoxyphenols inhibit RNR. Here, molecular docking and molecular dynamics simulations involving protein R2 suggest an inhibition mechanism for p-alkoxyphenols . A low energy binding pocket is identified in protein R2. The preferred configuration provides a structural basis explaining their specific binding to the Escherichia coli and mouse R2 proteins. Trp48 (E. coli numbering), on the electron transfer pathway, is involved in the interactions with the inhibitors. The relative order of the binding energies calculated for the phenol derivatives to protein R2 is correlated with earlier experimental data on inhibition efficiency, in turn related to increasing size of the hydrophobic alkyl substituents. Using the configuration identified by molecular docking as a starting point for molecular dynamics simulations, we find that the p-allyloxyphenol interrupts the catalytic electron transfer pathway of the R2 protein by forming hydrogen bonds with Trp48 and Asp237, thus explaining the inhibitory activity of p-alkoxyphenols.     

Computer simulations of membrane protein folding: structure and dynamics

A lattice model of membrane proteins with a composite energy function is proposed to study their folding dynamics and native structures using Monte Carlo simulations. This model successfully predicts the seven helix bundle structure of sensory rhodopsin I by practicing a three-stage folding. Folding dynamics of a transmembrane segment into a helix is further investigated by varying the cooperativity in the formation of alpha helices for both random folding and assisted folding. The chain length dependence of the folding time of a hydrophobic segment to a helical state is studied for both free and anchored chains. An unusual length dependence in the folding time of anchored chains is observed.     

DNA condensation by TmHU studied by optical tweezers,AFM and molecular dynamics simulations

The compaction of DNA by the HU protein from Thermotoga maritima (TmHU) is analysed on a single-molecule level by the usage of an optical tweezers-assisted force clamp. The condensation reaction is investigated at forces between 2 and 40 pN applied to the ends of the DNA as well as in dependence on the TmHU concentration. At 2 and 5 pN, the DNA compaction down to 30% of the initial end-to-end distance takes place in two regimes. Increasing the force changes the progression of the reaction until almost nothing is observed at 40 pN. Based on the results of steered molecular dynamics simulations, the first regime of the length reduction is assigned to a primary level of DNA compaction by TmHU. The second one is supposed to correspond to the formation of higher levels of structural organisation. These findings are supported by results obtained by atomic force microscopy.     

The structure of a membrane-spanning polypeptide studied by molecular dynamics

We have performed a molecular dynamics simulation of a 46-residue segment of glycophorin which includes the hydrophobic membrane-spanning region of this protein. The presence of a membrane and of water is taken into account in a continuum approximation which makes use of phenomenological hydrophobic energies. The initial -helical conformation and the membrane incorporation of the hydrophobic segment remain stable for the length of the simulation which is 100 ps. Moreover, when the hydrophobic segment is partially shifted out of the membrane, it moves back into the membrane. Superimposed on these deterministic effects one also observes thermal fluctuations in the form of bending and tilting of the membrane-spanning helix.     

Refinement of comparative models of protein structure by using multicanonical molecular dynamics simulations

Comparative modelling is a powerful method that easily predicts a considerably accurate structure of a protein by using a template structure having a similar amino-acid sequence to the target protein. However, in the region where the amino-acid sequence is different between the target and the template, the predicted structure remains unreliable. In such a case, the model has to be refined. In the present study, we explored the possibility of a molecular dynamics-based method, using the human SAP Src Homology 2 (SH2) domain as the modelling target. The multicanonical method was used to alleviate the multiple-minima problem and the generalised Born/surface area model was used to reduce the computational cost. In addition, position restraints were imposed on the atoms in the reliable regions to avoid unnecessary conformational sampling. We analyzed the conformational distribution of the ligand-recognition loop of the domain and found that the most populated conformational clusters in the ensemble of the model agreed well with one of the two major clusters in the ensemble of the reference simulation starting from the crystal structure. This demonstrates that the current refinement method can significantly improve the accuracy of an unreliable region in a comparative model.     

Thermostability of protein studied by molecular dynamics simulation

The thermostability of protein thermostable cathechol 2,3-dixoygenase (TC23O) has been studied by the parallel molecular dynamics simulations. By analysis of the exponent beta, which is related to the scattering spectrum and constant-pressure heat capacity Cp, we reveal the respective contribution of a specific residue 228 proline; a specific salt bridge, Lys188N-Glu291OE1; four ions; and a different water environment to the thermostability of TC23O. The dynamic transition temperature of the mutants, Pro228Ser and Glu291Gly of the TC23O, was decreased about 10 degrees C and 19 degrees C respectively. The displacement of the four ions had no significant effect on the thermostability of TC23O. Water affects the thermostability by influencing the changes of accessible conformation to a certain extent. All these results agree with the known experimental results.     

Sequence-dependent DNA deformability studied using molecular dynamics simulations

Proteins recognize specific DNA sequences not only through direct contact between amino acids and bases, but also indirectly based on the sequence-dependent conformation and deformability of the DNA (indirect readout). We used molecular dynamics simulations to analyze the sequence-dependent DNA conformations of all 136 possible tetrameric sequences sandwiched between CGCG sequences. The deformability of dimeric steps obtained by the simulations is consistent with that by the crystal structures. The simulation results further showed that the conformation and deformability of the tetramers can highly depend on the flanking base pairs. The conformations of xATx tetramers show the most rigidity and are not affected by the flanking base pairs and the xYRx show by contrast the greatest flexibility and change their conformations depending on the base pairs at both ends, suggesting tetramers with the same central dimer can show different deformabilities. These results suggest that analysis of dimeric steps alone may overlook some conformational features of DNA and provide insight into the mechanism of indirect readout during protein–DNA recognition. Moreover, the sequence dependence of DNA conformation and deformability may be used to estimate the contribution of indirect readout to the specificity of protein–DNA recognition as well as nucleosome positioning and large-scale behavior of nucleic acids.     

Mechanically induced titin kinase activation studied by force-probe molecular dynamics simulations

The conversion of mechanical stress into a biochemical signal in a muscle cell requires a force sensor. Titin kinase, the catalytic domain of the elastic muscle protein titin, has been suggested as a candidate. Its activation requires major conformational changes resulting in the exposure of its active site. Here, force-probe molecular dynamics simulations were used to obtain insight into the tension-induced activation mechanism. We find evidence for a sequential mechanically induced opening of the catalytic site without complete domain unfolding. Our results suggest the rupture of two terminal beta-sheets as the primary unfolding steps. The low force resistance of the C-terminal relative to the N-terminal beta-sheet is attributed to their different geometry. A subsequent rearrangement of the autoinhibitory tail is seen to lead to the exposure of the active site, as is required for titin kinase activity. These results support the hypothesis of titin kinase as a force sensor.     

Efficiency of the monofunctionalized C60 fullerenes as membrane targeting agents studied by all-atom molecular dynamics simulations

Abstract

Transmembrane translocation of C₆₀ fullerenes functionalized by the single amino-derivative in neutral and charged forms was studies by extensive all-atom molecular dynamics simulations. It is shown that these complexes exhibit very strong affinity to the membrane core, but their spontaneous translocation through the membrane is not possible at practical time scale. In contrast, free amino derivatives translocate through the membrane much easier than their complexes with fullerenes, but do not have pronounced affinity to the membrane interior. Our results suggest that monofunctionalized C₆₀ could be extremely efficient membrane targeting agents, which facilitate accumulation of the water-soluble compounds in the hydrophobic core of lipid bilayer.     

Computer simulations of protein folding by targeted molecular dynamics

We have performed 128 folding and 45 unfolding molecular dynamics runs of chymotrypsin inhibitor 2 (CI2) with an implicit solvation model for a total simulation time of 0.4 microseconds. Folding requires that the three-dimensional structure of the native state is known. It was simulated at 300 K by supplementing the force field with a harmonic restraint which acts on the root-mean-square deviation and allows to decrease the distance to the target conformation. High temperature and/or the harmonic restraint were used to induce unfolding. Of the 62 folding simulations started from random conformations, 31 reached the native structure, while the success rate was 83% for the 66 trajectories which began from conformations unfolded by high-temperature dynamics. A funnel-like energy landscape is observed for unfolding at 475 K, while the unfolding runs at 300 K and 375 K as well as most of the folding trajectories have an almost flat energy landscape for conformations with less than about 50% of native contacts formed. The sequence of events, i.e., secondary and tertiary structure formation, is similar in all folding and unfolding simulations, despite the diversity of the pathways. Previous unfolding simulations of CI2 performed with different force fields showed a similar sequence of events. These results suggest that the topology of the native state plays an important role in the folding process.     

Automated builder and database of protein/membrane complexes for molecular dynamics simulations

Molecular dynamics simulations of membrane proteins have provided deeper insights into their functions and interactions with surrounding environments at the atomic level. However, compared to solvation of globular proteins, building a realistic protein/membrane complex is still challenging and requires considerable experience with simulation software. Membrane Builder in the CHARMM-GUI website (http://www.charmm-gui.org) helps users to build such a complex system using a web browser with a graphical user interface. Through a generalized and automated building process including system size determination as well as generation of lipid bilayer, pore water, bulk water, and ions, a realistic membrane system with virtually any kinds and shapes of membrane proteins can be generated in 5 minutes to 2 hours depending on the system size. Default values that were elaborated and tested extensively are given in each step to provide reasonable options and starting points for both non-expert and expert users. The efficacy of Membrane Builder is illustrated by its applications to 12 transmembrane and 3 interfacial membrane proteins, whose fully equilibrated systems with three different types of lipid molecules (DMPC, DPPC, and POPC) and two types of system shapes (rectangular and hexagonal) are freely available on the CHARMM-GUI website. One of the most significant advantages of using the web environment is that, if a problem is found, users can go back and re-generate the whole system again before quitting the browser. Therefore, Membrane Builder provides the intuitive and easy way to build and simulate the biologically important membrane system.     

Proton transport across transient single-file water pores in a lipid membrane studied by molecular dynamics simulations.

To test the hypothesis that water pores in a lipid membrane mediate the proton transport, molecular dynamic simulations of a phospholipid membrane, in which the formation of a water pore is induced, are reported. The probability density of such a pore in the membrane was obtained from the free energy of formation of the pore, which was computed from the average force needed to constrain the pore in the membrane. It was found that the free energy of a single file of water molecules spanning the bilayer is 108(+/-10) kJ/mol. From unconstrained molecular dynamic simulations it was further deduced that the nature of the pore is very transient, with a mean lifetime of a few picoseconds. The orientations of water molecules within the pore were also studied, and the spontaneous translocation of a turning defect was observed. The combined data allowed a permeability coefficient for proton permeation across the membrane to be computed, assuming that a suitable orientation of the water molecules in the pore allows protons to permeate the membrane relatively fast by means of a wirelike conductance mechanism. The computed value fits the experimental data only if it is assumed that the entry of the proton into the pore is not rate limiting.     

Folding-unfolding of goat alpha-lactalbumin studied by stopped-flow circular dichroism and molecular dynamics simulations

Folding reaction of goat alpha-lactalbumin has been studied by stopped-flow circular dichroism and molecular dynamics simulations. The effects of four single mutations and a double mutation on the stability of the protein under a native condition were studied. The mutations were introduced into residues located at a hydrophobic core in the alpha-domain of the molecule. Here we show that an amino acid substitution (T29I) increases the native-state stability of goat alpha-lactalbumin against the guanidine hydrochloride-induced unfolding by 3.5 kcal/mol. Kinetic refolding and unfolding of wild-type and mutant goat alpha-lactalbumin measured by stopped-flow circular dichroism showed that the local structure around the Thr29 side chain was not constructed in the transition state of the folding reaction. To characterize the local structural change around the Thr29 side chain to an atomic level of resolution, we performed high-temperature (at 400 K and 600 K) molecular dynamics simulations and studied the structural change at an initial stage of unfolding observed in the simulation trajectories. The Thr29 portion of the molecule experienced structural disruption accompanied with the loss of inter-residue contacts and with the water molecule penetration in the 400-K simulation as well as in four of the six 600-K simulations. Disruption of the N-terminal portion was also observed and was consistent with the results of kinetic refolding/unfolding experiments shown in our previous report.