Analyzing the Molecular Basis of Enzyme Stability in Ethanol/Water Mixtures Using Molecular Dynamics Simulations

One of the drawbacks of nonaqueous enzymology is the fact that enzymes tend to be less stable in organic solvents than in water. There are, however, some enzymes that display very high stabilities in nonaqueous media. In order to take full advantage of the use of nonaqueous solvents in enzyme catalysis, it is essential to elucidate the molecular basis of enzyme stability in these media. Toward this end, we performed μs-long molecular dynamics simulations using two homologous proteases, pseudolysin, and thermolysin, which are known to have considerably different stabilities in solutions containing ethanol. The analysis of the simulations indicates that pseudolysin is more stable than thermolysin in ethanol/water mixtures and that the disulfide bridge between C30 and C58 is important for the stability of the former enzyme, which is consistent with previous experimental observations., Our results indicate that thermolysin has a higher tendency to interact with ethanol molecules (especially through van der Waals contacts) than pseudolysin, which can lead to the disruption of intraprotein hydrophobic interactions and ultimately result in protein unfolding. In the absence of the C30–C58 disulfide bridge, pseudolysin undergoes larger conformational changes, becoming more open and more permeable to ethanol molecules which accumulate in its interior and form hydrophobic interactions with the enzyme, destroying its structure. Our observations are not only in good agreement with several previous experimental findings on the stability of the enzymes studied in ethanol/water mixtures but also give an insight on the molecular determinants of this stability. Our findings may, therefore, be useful in the rational development of enzymes with increased stability in these media

Structural Determinants for the Membrane Insertion of the Transmembrane Peptide of Hemagglutinin from Influenza Virus

Membrane fusion is a process involved in a high range of biological functions, going from viral infections to neurotransmitter release. Fusogenic proteins increase the slow rate of fusion by coupling energetically downhill conformational changes of the protein to the kinetically unfavorable fusion of the membrane lipid bilayers. Hemagglutinin is an example of a fusogenic protein, which promotes the fusion of the membrane of the influenza virus with the membrane of the target cell. The N-terminus of the HA2 subunit of this protein contains a fusion domain described to act as a destabilizer of the target membrane bilayers, leading eventually to a full fusion of the two membranes. On the other hand, the C-terminus of the same subunit contains a helical transmembrane domain which was initially described to act as the anchor of the protein to the membrane of the virus. However, in recent years the study of this peptide segment has been gaining more attention since it has also been described to be involved in the membrane fusion process. Yet, the structural characterization of the interaction of such a protein domain with membrane lipids is still very limited. Therefore, in this work, we present a study of this transmembrane peptide domain in the presence of DMPC membrane bilayers, and we evaluate the effect of several mutations, and the effect of peptide oligomerization in this interaction process. Our results allowed us to identify and confirm amino acid residue motifs that seem to regulate the interaction between the segment peptide and membrane bilayers. Besides these sequence requirements, we have also identified length and tilt requirements that ultimately contribute to the hydrophobic matching between the peptide and the membrane. Additionally, we looked at the association of several transmembrane peptide segments and evaluated their direct interaction and stability inside a membrane bilayer. From our results we could conclude that three independent TM peptide segments arrange themselves in a parallel arrangement, very similarly to what is observed for the C-terminal regions of the hemagglutinin crystallographic structure of the protein, to where the segments are attached

Constant-pH MD Simulations of an Oleic Acid Bilayer

Oleic acid is a simple molecule with an aliphatic chain and a carboxylic group whose ionization and, consequently, intermolecular interactions are strongly dependent on the solution pH. The titration curve of these molecules was already obtained using different experimental methods, which have shown the lipid bilayer assemblies to be stable between pH 7.0 and 9.0. In this work, we take advantage of our recent implementations of periodic boundary conditions in Poisson−Boltzmann calculations and ionic strength treatment in simulations of charged lipid bilayers, and we studied the ionization dependent behavior of an oleic acid bilayer using a new extension of the stochastic titration constant-pH MD method. With this new approach, we obtained titration curves that are in good agreement with the experimental data. Also, we were able to estimate the slope of the titration curve from charge fluctuations, which is an important test of thermodynamic consistency for the sampling in a constant-pH MD method. The simulations were performed for ionizations up to 50%, because an experimentally observed macroscopic transition to micelles occurs above this value. As previously seen for a binary mixture of a zwitterionic and an anionic lipid, we were able to reproduce experimental results with simulation boxes usually far from neutrality. This observation further supports the idea that a charged membrane strongly influences the ion distribution in its vicinity and that neutrality is achieved significantly far from the bilayer surface. The good results obtained with this extension of the stochastic titration constant-pH MD method strongly supports its usefulness to sample the coupling between configuration and protonation in these types of biophysical systems. This method stands now as a powerful tool to study more realistic lipid bilayers where pH can influence both the lipids and the solutes interacting with them

Unraveling the Conformational Determinants of Peptide Dendrimers Using Molecular Dynamics Simulations

Peptide dendrimers are synthetic tree-like molecules composed of amino acids. There are at least two kinds of preferential structural behaviors exhibited by these molecules, which acquire either compact or noncompact shapes. However, the key structural determinants of such behaviors remained, until now, unstudied. Herein, we conduct a comprehensive investigation of the structural determinants of peptide dendrimers by employing long molecular dynamics simulations to characterize an extended set of third generation dendrimers. Our results clearly show that a trade-off between electrostatic effects and hydrogen bond formation controls structure acquisition in these systems. Moreover, by selectively changing the dendrimers charge we are able to manipulate the exhibited compactness. In contrast, the length of branching residues does not seem to be a major structural determinant. Our results are in accordance with the most recent experimental evidence and shed some light on the key molecular level interactions controlling structure acquisition in these systems. Thus, the results presented constitute valuable insights that can contribute to the development of truly tailor-made dendritic systems

Protonation of DMPC in a Bilayer Environment Using a Linear Response Approximation

pH is a very important property, influencing all important biomolecules such as proteins, nucleic acids, and lipids. The effect of pH on proteins has been the subject of many computational works in recent years. However, the same has not been done for lipids, especially in their most biologically relevant environment: the bilayer. A reason for this is the inherent technical difficulty in dealing with this type of periodic systems. Here, we tackle this problem by developing a Poisson–Boltzmann-based method that takes in consideration the periodic boundary conditions of lipid bilayer patches. We used this approach with a linear response approximation to calculate the p<i>K</i>_a value of a DMPC molecule when diluted in zwitterionic lipids. Our results show that DMPC protonation only becomes relevant at quite low pH values (2–3). However, when it happens, it has a strong impact on lipid conformations, leading to significant heterogeneity in the membrane

Reversibility of Prion Misfolding: Insights from Constant-pH Molecular Dynamics Simulations

The prion protein (PrP) is the cause of a group of diseases known as transmissible spongiform encephalopathies (TSEs). Creutzfeldt–Jakob disease and bovine spongiform encephalopathy are examples of TSEs. Although the normal form of PrP (PrP^C) is monomeric and soluble, it can misfold into a pathogenic form (PrP^Sc) that has a high content of β-structure and can aggregate forming amyloid fibrils. The mechanism of conversion of PrP^C into PrP^Sc is not known but different triggers have been proposed. It can be catalyzed by a PrP^Sc sample, or it can be induced by an external factor, such as low pH. The pH effect on the structure of PrP was recently studied by computational methods [Campos et al. <i>J. Phys. Chem. B</i> <b>2010</b>, <i>114</i>, 12692–12700], and an evident trend of loss of helical structure was observed with pH decrease, together with a gain of β-structures. In particular, one simulation at pH 2 showed an evident misfolding transition. The main goal of the present work was to study the effects of a change in pH to 7 in several transient conformations of this simulation, in order to draw some conclusions about the reversibility of PrP misfolding. Although the most significant effect caused by the change of pH to 7 was a global stabilization of the protein structure, we could also observe that some conformational transitions induced by pH 2 were reversible in many of our simulations, namely those started from the early moments of the misfolding transition. This observation is in good agreement with experiments showing that, even at pH as low as 1.7, it is possible to revert the misfolding process [Bjorndahl et al. <i>Biochemistry</i> <b>2011</b>, <i>50</i>, 1162–1173]

Structural Effects of pH and Deacylation on Surfactant Protein C in an Organic Solvent Mixture: A Constant-pH MD Study

The pulmonary surfactant protein C (SP-C) is a small highly hydrophobic protein that adopts a mainly helical structure while associated with the membrane but misfolds into a β-rich metastable structure upon deacylation, membrane dissociation, and exposure to the neutral pH of the aqueous alveolar subphase, eventually leading to the formation of amyloid aggregates associated with pulmonary alveolar proteinosis. The present constant-pH MD study of the acylated and deacylated isoforms of SP-C in a chloroform/methanol/water mixture, often used to mimic the membrane environment, shows that the loss of the acyl groups has a structural destabilizing effect and that the increase of pH promotes intraprotein contacts which contribute to the loss of helical structure in solution. These contacts result from the poor solvation of charged groups by the solvent mixture, which exhibits a limited membrane-mimetic character. Although a single SP-C molecule was used in the simulations, we propose that analogous intermolecular interactions may play a role in the early stages of the protein misfolding and aggregation in this mixture

Interaction of Counterions with Subtilisin in Acetonitrile: Insights from Molecular Dynamics Simulations

A recent X-ray structure has enabled the location of chloride and cesium ions on the surface of subtilisin Carlsberg in acetonitrile soaked crystals. To complement the previous study and analyze the system in solution, molecular dynamics (MD) simulations, in acetonitrile, were performed using this structure. Additionally, Cl^– and Cs⁺ ions were docked on the protein surface and this system was also simulated. Our results indicate that chloride ions tend to stay close to the protein, whereas cesium ions frequently migrate to the solvent. The distribution of the ions around the enzyme surface is not strongly biased by their initial locations. Replacing cesium by sodium ions showed that the distribution of the two cations is similar, indicating that Cs⁺ can be used to find the binding sites of cations like Na⁺ and K⁺, which, unlike Cs⁺, have physiological and biotechnological roles. The Na⁺Cl^– is more stable than the Cs⁺Cl^– ion pair, decreasing the probability of interaction between Cl^– and subtilisin. The comparison of water and acetonitrile simulations indicates that the solvent influences the distribution of the ions. This work provides an extensive theoretical analysis of the interaction between ions and the model enzyme subtilisin in a nonaqueous medium

Treatment of Ionic Strength in Biomolecular Simulations of Charged Lipid Bilayers

Biological membranes are complex systems that have recently attracted a significant scientific interest. Due to the presence of many different anionic lipids, these membranes are usually negatively charged and sensitive to pH. The protonation states of lipids and the ion distribution close to the bilayer are two of the main challenges in biomolecular simulations of these systems. These two problems have been circumvented by using ionized (deprotonated) anionic lipids and enough counterions to preserve the electroneutrality. In this work, we propose a method based on the Poisson–Boltzmann equation to estimate the counterion and co-ion concentration close to a lipid bilayer that avoids the need for neutrality at this microscopic level. The estimated number of ions was tested in molecular dynamics simulations of a 25% DMPA/DMPC lipid bilayer at different ionization levels. Our results show that the system neutralization represents an overestimation of the number of counterions. Consequently, the resulting lipid bilayer becomes too ordered and practically insensitive to ionization. On the other hand, our proposed approach is able to correctly model the ionization dependent isothermal phase transition of the bilayer observed experimentally. Furthermore, our approach is not too computationally expensive and can easily be used to model diverse charged biomolecular systems in molecular dynamics simulations

Interaction of Counterions with Subtilisin in Acetonitrile: Insights from Molecular Dynamics Simulations

A recent X-ray structure has enabled the location of chloride and cesium ions on the surface of subtilisin Carlsberg in acetonitrile soaked crystals. To complement the previous study and analyze the system in solution, molecular dynamics (MD) simulations, in acetonitrile, were performed using this structure. Additionally, Cl^– and Cs⁺ ions were docked on the protein surface and this system was also simulated. Our results indicate that chloride ions tend to stay close to the protein, whereas cesium ions frequently migrate to the solvent. The distribution of the ions around the enzyme surface is not strongly biased by their initial locations. Replacing cesium by sodium ions showed that the distribution of the two cations is similar, indicating that Cs⁺ can be used to find the binding sites of cations like Na⁺ and K⁺, which, unlike Cs⁺, have physiological and biotechnological roles. The Na⁺Cl^– is more stable than the Cs⁺Cl^– ion pair, decreasing the probability of interaction between Cl^– and subtilisin. The comparison of water and acetonitrile simulations indicates that the solvent influences the distribution of the ions. This work provides an extensive theoretical analysis of the interaction between ions and the model enzyme subtilisin in a nonaqueous medium