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

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

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]

p<i>K</i>_a Values of Titrable Amino Acids at the Water/Membrane Interface

Peptides and proteins protonation equilibrium is strongly influenced by its surrounding media. Remarkably, until now, there have been no quantitative and systematic studies reporting the p<i>K</i>_a shifts in the common titrable amino acids upon lipid membrane insertion. Here, we applied our recently developed CpHMD-L method to calculate the p<i>K</i>_a values of titrable amino acid residues incorporated in Ala-based pentapeptides at the water/membrane interface. We observed that membrane insertion leads to desolvation and a clear stabilization of the neutral forms, and we quantified the increases/decreases of the p<i>K</i>_a values in the anionic/cationic residues along the membrane normal. This work highlights the importance of properly modeling the protonation equilibrium in peptides and proteins interacting with membranes using molecular dynamics simulations

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

Constant-pH MD Simulations of DMPA/DMPC Lipid Bilayers

Current constant-pH molecular dynamics (CpHMD) simulations provide a proper treatment of pH effects on the structure and dynamics of soluble biomolecules like peptides and proteins. However, addressing such effects on lipid membrane assemblies has remained problematic until now, despite the important role played by lipid ionization at physiological pH in a plethora of biological processes. Modeling (de)­protonation events in these systems requires a proper consideration of the physicochemical features of the membrane environment, including a sound treatment of solution ions. Here, we apply our recent CpHMD-L method to the study of pH effects on a 25% DMPA/DMPC bilayer membrane model, closely reproducing the correct lipid phases of this system, namely, gel–fluid coexistence at pH 4 and a fluid phase at pH 7. A significant transition is observed for the membrane ionization and mechanical properties at physiological pH, providing a molecular basis for the well-established role of phosphatidic acid (PA) as a key player in the regulation of many cellular events. Also, as reported experimentally, we observed pH-induced PA–PA lipid aggregation at acidic pH. By including the titration of anionic phospholipids, the current methodology makes possible to simulate lipid bilayers with increased realism. To the best of our knowledge, this is the first simulation study dealing with a continuous phospholipid bilayer with pH titration of all constituent lipids

ΔN6-N and ΔN6-I dimer conformations.

<p>Representative dimer conformations with number of intermolecular contacts equal to the mode (mode dimers) and tail (strongly-matched dimers) of the density histograms. Mode dimers of ΔN6-N (A) and ΔN6-I (B) at pH 7.2, ΔN6-N (C) and ΔN6-I (D) at pH 6.2. Strongly-matched dimers of ΔN6-N (E) and ΔN6-I (F) at pH 7.2, ΔN6-N (G) and ΔN6-I (H) at pH 6.2. The ΔN6-I mode dimers (and the strongly-matched dimers at pH 6.2) highlight the involvement of strand A of one of the monomers in dimerization and the intrinsic possibility of further oligomerization via the second monomer's solvent-exposed strand A. If the unstructured strands A of both monomers are involved in dimerization (as in the strongly-packed dimer at pH 7.2, F) further growth is still possible via the highly solvent-exposed and aromatic-rich DE-loops (highlighted in green). In ΔN6-N the AB-loop and the “unstructured” DE-loop (which is detached from the protein's core and exposed to the solvent) account for the possibility of further oligomerization. Other examples of ΔN6-I ‘mode’ dimers are reported in <b><a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003606#pcbi.1003606.s003" target="_blank">Fig. S3C</a></b> and <b><a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003606#pcbi.1003606.s003" target="_blank">Fig. S3D</a></b>. (I) Pictorial representation of monomers of ΔN6-I that were packed ‘by hand’ to indicate a possible oligomerization pattern leading to amyloid fibrils.</p

Molecular Details of INH‑C₁₀ Binding to <i>wt</i> KatG and Its S315T Mutant

Isoniazid (INH) is still one of the two most effective antitubercular drugs and is included in all recommended multitherapeutic regimens. Because of the increasing resistance of <i>Mycobacterium tuberculosis</i> to INH, mainly associated with mutations in the <i>katG</i> gene, new INH-based compounds have been proposed to circumvent this problem. In this work, we present a detailed comparative study of the molecular determinants of the interactions between <i>wt</i> KatG or its S315T mutant form and either INH or INH-C₁₀, a new acylated INH derivative. MD simulations were used to explore the conformational space of both proteins, and results indicate that the S315T mutation did not have a significant impact on the average size of the access tunnel in the vicinity of these residues. Our simulations also indicate that the steric hindrance role assigned to Asp137 is transient and that electrostatic changes can be important in understanding the enzyme activity data of mutations in KatG. Additionally, molecular docking studies were used to determine the preferred modes of binding of the two substrates. Upon mutation, the apparently less favored docking solution for reaction became the most abundant, suggesting that S315T mutation favors less optimal binding modes. Moreover, the aliphatic tail in INH-C₁₀ seems to bring the hydrazine group closer to the heme, thus favoring the apparent most reactive binding mode, regardless of the enzyme form. The ITC data is in agreement with our interpretation of the C₁₀ alkyl chain role and helped to rationalize the significantly lower experimental MIC value observed for INH-C₁₀. This compound seems to be able to counterbalance most of the conformational restrictions introduced by the mutation, which are thought to be responsible for the decrease in INH activity in the mutated strain. Therefore, INH-C₁₀ appears to be a very promising lead compound for drug development