Christophe Ramseyer

Insertion of Short Amino-Functionalized Single-Walled Carbon Nanotubes into Phospholipid Bilayer Occurs by Passive Diffusion

Carbon nanotubes have been proposed to be efficient nanovectors able to deliver genetic or therapeutic cargo into living cells. However, a direct evidence of the molecular mechanism of their translocation across cell membranes is still needed. Here, we report on an extensive computational study of short (5 nm length) pristine and functionalized single-walled carbon nanotubes uptake by phospholipid bilayer models using all-atom molecular dynamics simulations. Our data support the hypothesis of a direct translocation of the nanotubes through the phospholipid membrane. We find that insertion of neat nanotubes within the bilayer is a “nanoneedle” like process, which can often be divided in three consecutive steps: landing and floating, penetration of the lipid headgroup area and finally sliding into the membrane core. The presence of functional groups at moderate concentrations does not modify the overall scheme of diffusion mechanism, provided that their deprotonated state favors translocation through the lipid bilayer.

Affinity of C60 Neat Fullerenes with Membrane Proteins: A Computational Study on Potassium Channels

Most studies of the interactions of neat and functionalized fullerenes with cells have focused so far on their ability to cross the cell membrane envelopes. Membranes are, however, also host to a large number of proteins responsible for various cellular functions. Among these, ion channels are prominent components of the nervous system. Recently, it was shown that fullerenes may act as blockers or modulators of a variety of K+ channels. Here we use computer simulations to investigate the propensity of such nanocompounds to bind to K+ channels. Our results based on extensive atomistic molecular dynamics simulations reveal a variety of specific binding sites depending on the structure and properties of the channel. The corresponding binding free energies and putative mechanisms suggest that C60 may indeed effectively hinder the function of K+ channels and hence induce toxicity.

New bioinspired membrane made of a biological ion channel confined into the cylindrical nanopore of a solid-state polymer.

A hybrid nanoporous membrane made of a solid-state polymeric thin film in which an ion channel is confined is realized. The primary and extremely encouraging results obtained by confocal fluorescence spectroscopy and ion diffusion measurement demonstrate respectively that (i) the considered ion channel, that is, Gramicidin-A, can be confined selectively inside the nanopores and (ii) the ionic permeability of the membrane is enhanced. Atomistic molecular simulations are also reported and fruitfully compared to the experimental findings.

Numerical studies of the membrane fluorescent dyes dynamics in ground and excited states.

Fluorescence methods are widely used in studies of biological and model membranes. The dynamics of membrane fluorescent markers in their ground and excited electronic states and correlations with their molecular surrounding within the fully hydrated phospholipid bilayer are still not well understood. In the present work, Quantum Mechanical (QM) calculations and Molecular Dynamics (MD) simulations are used to characterize location and interactions of two membrane polarity probes (Prodan; 6-propionyl-2-dimethylaminonaphthalene and its derivative Laurdan; 2-dimethylamino-6-lauroylnaphthalene) with the dioleoylphosphatidylcholine (DOPC) lipid bilayer model. MD simulations with fluorophores in ground and excited states are found to be a useful tool to analyze the fluorescent dye dynamics and their immediate vicinity. The results of QM calculations and MD simulations are in excellent agreement with available experimental data. The calculation shows that the two amphiphilic dyes initially placed in bulk water diffuse within 10 ns towards their final location in the lipid bilayer. Analysis of solvent relaxation process in the aqueous phase occurs on the picoseconds timescale whereas it takes nanoseconds at the lipid/water interface. Four different relaxation time constants, corresponding to different relaxation processes, where observed when the dyes were embedded into the membrane.

How do functionalized carbon nanotubes land on, bind to and pierce through model and plasma membranes†

Study of the mechanisms understanding how chemically functionalized carbon nanotubes internalize into mammalian cells is important in view of their design as new tools for therapeutic and diagnostic applications. The initial contact between the nanotube and the cell membrane allows elucidation of the types of interaction that are occurring and the contribution from the types of functional groups at the nanotube surface. Here we offer a combination of experimental and theoretical evidence of the initial phases of interaction between functionalized carbon nanotubes with model and cellular membranes. Both experimental and theoretical data reveal the critical parameters to determine direct translocation of the nanotubes through the membrane into the cytoplasm as a result of three distinct processes that can be summarized as landing, piercing and uptake.

Targeted molecular dynamics of an open-state KcsA channel

Pore opening of KcsA channel is studied using targeted molecular dynamics simulations. Conformational changes of the protein are determined, starting from the crystallized refined 2.0 A structure (pdb 1K4C) determined in x-ray experiments and arriving to the open-state structure constructed on the basis of electron paramagnetic resonance spectroscopic data (pdb 1JQ1). Our results corroborate the essential role played by the terminal residues located on the transmembrane helices M2 which were not taken into account at that time. The aperture mechanism of the channel appears to be ziplike. A small constraint (approximately equal to 5 x 10(-2) kcal mol(-1) A(-2) per C(alpha)) applied to the terminal residues located on the intracellular side is sufficient to initialize the pore opening at the innermost part of the gate, but additional constraint must be applied to definitely complete the pore aperture. The open structure is proved to be a metastable state since releasing the constraint leads to another relaxed open conformation which seems to reach stability.

Uptake and Translocation Mechanisms of Cationic Amino Derivatives Functionalized on Pristine C60 by Lipid Membranes: A Molecular Dynamics Simulation Study

Bioactive molecules, cationic peptides among them, are nowadays well-recognized in modern pharmacology for their drug potential. However, they usually suffer from poor translocation across cell membranes, and specific drug carriers should be designed to circumvent this problem. In the present study, the uptake mechanism of fullerene bearing cationic ammonium groups by membranes modeled as lipid bilayers is investigated using extensive molecular dynamics simulations and free-energy calculations. Three main results issued from this work can be drawn. First, the fullerene core appears to be a good drug vector since it greatly enhances the uptake of the cationic groups by the membrane. Second, we show that the amino derivatives should be deprotonated at the lipid headgroup level in order to fully translocate the membrane by passive diffusion. Finally, the fullerenes bearing too many cationic groups display mostly a hydrophilic character; thus, the lipophilic fullerene core is not anymore effective as an insertion enhancer. Therefore, the lipid bilayer appears to be very selective with respect to the amount of amino groups conjugated with C(60).

Cholesterol Induces Uneven Curvature of Asymmetric Lipid Bilayers

A remarkable flexibility is observed in biological membranes, which allows them to form the structures of different curvatures. We addressed the question of intrinsic ability of phospholipid membranes to form highly curved structures and the role of cholesterol in this process. The distribution of cholesterol in the highly curved asymmetric DOPC/DOPS lipid bilayer was investigated by the coarse-grained molecular dynamics simulations in the membrane patches with large aspect ratio. It is shown that cholesterol induces uneven membrane curvature promoting the formation of extended flattened regions of the membrane interleaved by sharp bends. It is shown that the affinity of cholesterol to anionic DOPS or neutral DOPC lipids is curvature dependent. The cholesterol prefers DOPS to DOPC in either planar or highly curved parts of the membrane. In contrast, in the narrow interval of moderate membrane curvatures this preference is inverted. Our data suggest that there is a complex self-consistent interplay between the membrane curvature and cholesterol distribution in the asymmetric lipid bilayers. The suggested new function of cholesterol may have a biological relevance.

About the structural role of disulfide bridges in serum albumins: Evidence from protein simulated unfolding†

The role of the 17 disulfide (S-S) bridges in preserving the native conformation of human serum albumin (HSA) is investigated by performing classical molecular dynamics (MD) simulations on protein structures with intact and, respectively, reduced S-S bridges. The thermal unfolding simulations predict a clear destabilization of the protein secondary structure upon reduction of the S-S bridges as well as a significant distortion of the tertiary structure that is revealed by the changes in the protein native contacts fraction. The effect of the S-S bridges reduction on the protein compactness was tested by calculating Gibbs free energy profiles with respect to the protein gyration radius. The theoretical results obtained using the OPLS-AA and the AMBER ff03 force fields are in agreement with the available experimental data. Beyond the validation of the simulation method, the results here reported provide new insights into the mechanism of the protein reductive/oxidative unfolding/folding processes. It is predicted that in the native conformation of the protein, the thiol (-SH) groups belonging to the same reduced S-S bridge are located in potential wells that maintain them in contact. The -SH pairs can be dispatched by specific conformational transitions of the peptide chain located in the neighborhood of the cysteine residues.

Will C-Laurdan dethrone Laurdan in fluorescent solvent relaxation techniques for lipid membrane studies?

Development of fluorescence methods involves the necessity of understanding the fluorescent probes behavior in their ground and excited states. In the case of biological membranes, the position of the dyes in the lipid bilayer and their response after excitation are necessary parameters to correctly analyze the experiments. In the present work, we focus on two fluorescent markers, Laurdan (6-lauroyl-2-(N,N-dimethylamino)naphthalene) and its derivative C-Laurdan (6-dodecanoyl-2-[N-methyl-N-(carboxymethyl)amino]naphthalene), recently proposed for lipid raft visualization [Kim, H. M.; et al. ChemBioChem 2007, 8, 553]. C-Laurdan, by the presence of an additional carboxyl group, has an advantage over Laurdan since it has a higher sensitivity to the membrane polarity at the lipid headgroup region and a higher water solubility. This theoretical study, based on quantum mechanical (QM) and molecular dynamics (MD) simulations in a fully hydrated lipid membrane model, compare the equilibrium and dynamic properties of both probes taking into account their fluorescence lifetimes. C-Laurdan is found to be more stable than Laurdan in the headgroup region of the membrane and also much more aligned with the lipids. This study suggests that, besides the lipid raft imaging, the C-Laurdan marker can considerably extend the capabilities of fluorescent solvent relaxation method.