Ionizable Cationic Lipids and Helper Lipids Synergistically Contribute to RNA Packing and Protection in Lipid-Based Nanomaterials.

Lipid-based nanomaterials are used as a common delivery vehicle for RNA therapeutics. They typically include a formulation containing ionizable cationic lipids, cholesterol, phospholipids, and a small molar fraction of PEGylated lipids. The ionizable cationic lipids are considered a crucial element of the formulation for the way they mediate interactions with the anionic RNA as a function of pH.

Structural basis of p53 inactivation by cavity-creating cancer mutations and its implications for the development of mutant p53 reactivators.

The cavity-creating p53 cancer mutation Y220C is an ideal paradigm for developing small-molecule drugs based on protein stabilization. Here, we have systematically analyzed the structural and stability effects of all oncogenic Tyr-to-Cys mutations (Y126C, Y163C, Y205C, Y220C, Y234C, and Y236C) in the p53 DNA-binding domain (DBD). They were all highly destabilizing, drastically lowering the melting temperature of the protein by 8-17 °C.

Computational approaches to lipid-based nucleic acid delivery systems.

Nucleic acid-based therapies have shown enormous effectiveness as vaccines against the recent COVID19 pandemics and hold great promises in the fight of a broad spectrum of diseases ranging from viral infections to cancer up to genetically transmitted pathologies. Due to their highly degradable polyanionic nature, nucleic acids need to be packed in sophisticate delivery vehicles which compact them up, protect them from early degradation and help delivery them to the right tissue/cells. Lipid-based nanoparticles (LNP) represent, at present, the main solution for nucleic acid delivery.

pH-Dependent Behavior of Ionizable Cationic Lipids in mRNA-Carrying Lipoplexes Investigated by Molecular Dynamics Simulations.

Lipid-based nanoparticles and lipoplexes containing ionizable lipids are among the most successful nanocarriers for mRNA-based therapies. The molecular structure of these assemblies is still not fully understood, as well as the role played by the ionizable lipids. SAXS experiments have shown that lipoplexes including the ionizable lipid 2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), under specific conditions, have a lamellar structure, where lipid bilayers are separated by mRNA-rich layers, with an overall spacing between 6.

Data Reweighting in Metadynamics Simulations.

The data collected along a metadynamics simulation can be used to recover information about the underlying unbiased system by means of a reweighting procedure. Here, we analyze the behavior of several reweighting techniques in terms of the quality of the reconstruction of the underlying unbiased free energy landscape in the early stages of the simulation and propose a simple reweighting scheme that we relate to the other techniques. We then show that the free energy landscape reconstructed from reweighted data can be more accurate than the negative bias potential depending on the reweighting technique, the stage of the simulation, and the adoption of well-tempered or standard metadynamics.

Targeting Cavity-Creating p53 Cancer Mutations with Small-Molecule Stabilizers: the Y220X Paradigm.

We have previously shown that the thermolabile, cavity-creating p53 cancer mutant Y220C can be reactivated by small-molecule stabilizers. In our ongoing efforts to unearth druggable variants of the p53 mutome, we have now analyzed the effects of other cancer-associated mutations at codon 220 on the structure, stability, and dynamics of the p53 DNA-binding domain (DBD). We found that the oncogenic Y220H, Y220N, and Y220S mutations are also highly destabilizing, suggesting that they are largely unfolded under physiological conditions.

Poly-sarcosine and Poly(Ethylene-Glycol) Interactions with Proteins Investigated Using Molecular Dynamics Simulations.

Nanoparticles coated with hydrophilic polymers often show a reduction in unspecific interactions with the biological environment, which improves their biocompatibility. The molecular determinants of this reduction are not very well understood yet, and their knowledge may help improving nanoparticle design. Here we address, using molecular dynamics simulations, the interactions of human serum albumin, the most abundant serum protein, with two promising hydrophilic polymers used for the coating of therapeutic nanoparticles, poly(ethylene-glycol) and poly-sarcosine.

Protein corona composition of poly(ethylene glycol)- and poly(phosphoester)-coated nanoparticles correlates strongly with the amino acid composition of the protein surface.

Extensive molecular dynamics simulations reveal that the interactions between proteins and poly(ethylene glycol) (PEG) can be described in terms of the surface composition of the proteins. PEG molecules accumulate around non-polar residues while avoiding the polar ones. A solvent-accessible-surface-area model of protein adsorption accurately fits a large set of data on the composition of the protein corona of poly(ethylene glycol)- and poly(phosphoester)-coated nanoparticles recently obtained by label-free proteomic mass spectrometry.

Molecular Dynamics Simulations of the Initial Adsorption Stages of Fibrinogen on Mica and Graphite Surfaces.

Fibrinogen, a blood glycoprotein of vertebrates, plays an essential role in blood clotting by polymerizing into fibrin when activated. Upon adsorption on material surfaces, it also contributes to determine their biocompatibility and has been implicated in the onset of thrombosis and inflammation at medical implants. Here we present the first fully atomistic simulations of the initial stages of the adsorption process of fibrinogen on mica and graphite surfaces.

The Internal Dynamics of Fibrinogen and Its Implications for Coagulation and Adsorption.

Fibrinogen is a serum multi-chain protein which, when activated, aggregates to form fibrin, one of the main components of a blood clot. Fibrinolysis controls blood clot dissolution through the action of the enzyme plasmin, which cleaves fibrin at specific locations. Although the main biochemical factors involved in fibrin formation and lysis have been identified, a clear mechanistic picture of how these processes take place is not available yet.

Simulations and experiments in protein folding.

The interplay between simulations and experiments of protein folding has largely contributed to the elucidation of many important aspects of the phenomenon. In this chapter, I briefly describe the experiments which provide information on the kinetics of the protein folding process, and help to characterize the folding transition state. Then, I show how to probe the kinetics of protein folding using molecular dynamics simulations, how to compare the simulations with the experiments and how to help and rationalize the latter, ultimately offering a molecular picture of the process.

Effects of ligand binding on the mechanical properties of ankyrin repeat protein gankyrin.

Ankyrin repeat proteins are elastic materials that unfold and refold sequentially, repeat by repeat, under force. Herein we use atomistic molecular dynamics to compare the mechanical properties of the 7-ankyrin-repeat oncoprotein Gankyrin in isolation and in complex with its binding partner S6-C. We show that the bound S6-C greatly increases the resistance of Gankyrin to mechanical stress.

Combination of Markov state models and kinetic networks for the analysis of molecular dynamics simulations of peptide folding.

Atomistic molecular dynamics simulations of the TZ1 beta-hairpin peptide have been carried out using an implicit model for the solvent. The trajectories have been analyzed using a Markov state model defined on the projections along two significant observables and a kinetic network approach. The Markov state model allowed for an unbiased identification of the metastable states of the system, and provided the basis for commitment probability calculations performed on the kinetic network.

Residue-resolved stability of full-consensus ankyrin repeat proteins probed by NMR.

We investigated the stability determinants and the unfolding characteristics of full-consensus designed ankyrin repeat proteins (DARPins) by NMR. Despite the repeating sequence motifs, the resonances could be fully assigned using (2)H,(15)N,(13)C triple-labeled proteins. To remove further ambiguities, we attached paramagnetic spin labels to either end of these elongated proteins, which attenuate the resonances of the spatially closest residues.

Mechanical unfolding of an ankyrin repeat protein.

Ankryin repeat proteins comprise tandem arrays of a 33-residue, predominantly alpha-helical motif that stacks roughly linearly to produce elongated and superhelical structures. They function as scaffolds mediating a diverse range of protein-protein interactions, and some have been proposed to play a role in mechanical signal transduction processes in the cell. Here we use atomic force microscopy and molecular-dynamics simulations to investigate the natural 7-ankyrin repeat protein gankyrin.

Toward the rational design of p53-stabilizing drugs: probing the surface of the oncogenic Y220C mutant.

The p53 cancer mutation Y220C induces formation of a cavity on the protein's surface that can accommodate stabilizing small molecules. We combined fragment screening and molecular dynamics to assess the druggability of p53-Y220C and map ligand interaction sites within the mutational cavity. Elucidation of the binding mode of fragment hits by crystallography yielded a clear picture of how a drug might dock in the cavity.

Multiple conformations of full-length p53 detected with single-molecule fluorescence resonance energy transfer.

The tumor suppressor p53 is a member of the emerging class of proteins that have both folded and intrinsically disordered domains, which are a challenge to structural biology. Its N-terminal domain (NTD) is linked to a folded core domain, which has a disordered link to the folded tetramerization domain, which is followed by a disordered C-terminal domain. The quaternary structure of human p53 has been solved by a combination of NMR spectroscopy, electron microscopy, and small-angle X-ray scattering (SAXS), and the NTD ensemble structure has been solved by NMR and SAXS.

Downhill versus barrier-limited folding of BBL 3. Heterogeneity of the native state of the BBL peripheral subunit binding domain and its implications for folding mechanisms.

Protein folding studies are generally predicated on Anfinsen's dogma that there is a unique native state of a protein. However, this is not always the case. NMR measurements of BBL, for example, find a decrease in helicity of helix 2 surrounding His166 on its protonation, which, with other experimental data, suggests that the native state can occupy two or more conformations.

Structure of human MDM4 N-terminal domain bound to a single-domain antibody.

The N-terminal domain of MDM4 binds to the N-terminal transactivation domain of the tumor suppressor p53 and is an important negative regulator of its transactivation activity. As such, inhibition of the binding of MDM4 to p53 is a target for anticancer therapy. The protein has not been crystallized satisfactorily for structural studies without the addition of an N-terminal p53 peptide.

High temperature unfolding simulations of the TRPZ1 peptide.

We report high temperature molecular dynamics simulations of the unfolding of the TRPZ1 peptide using an explicit model for the solvent. The system has been simulated for a total of 6 mus with 100-ns minimal continuous stretches of trajectory. The populated states along the simulations are identified by monitoring multiple observables, probing both the structure and the flexibility of the conformations.

Fluorescence resonance energy transfer analysis of the folding pathway of Engrailed Homeodomain.

The Engrailed Homeodomain folds on the microsecond time scale via an intermediate that is experimentally well characterised using structural Engrailed-Homeodomain mimics. Here, we analysed directly the changes in distance between key residues during the kinetics of unfolding and at equilibrium using fluorescence resonance energy transfer (FRET). Trp was the donor and 5-(((acetylamino)ethyl)amino) naphthalene-1-sulphate, the acceptor, substituted in positions that caused little change in stability.

Folding and unfolding mechanism of highly stable full-consensus ankyrin repeat proteins.

Full-consensus designed ankyrin repeat proteins were designed with one to six identical repeats flanked by capping repeats. These proteins express well in Escherichia coli as soluble monomers. Compared to our previously described designed ankyrin repeat protein library, randomized positions have now been fixed according to sequence statistics and structural considerations.

Characterization and further stabilization of designed ankyrin repeat proteins by combining molecular dynamics simulations and experiments.

Multiple molecular dynamics simulations with explicit solvent at room temperature and at 400 K were carried out to characterize designed ankyrin repeat (AR) proteins with full-consensus repeats. Using proteins with one to five repeats, the stability of the native structure was found to increase with the number of repeats. The C-terminal capping repeat, originating from the natural guanine-adenine-binding protein, was observed to denature first in almost all high-temperature simulations.

Wordom: a program for efficient analysis of molecular dynamics simulations.

Wordom is a versatile program for manipulation of molecular dynamics trajectories and efficient analysis of simulations. Original tools in Wordom include a procedure to evaluate significance of sampling for principal component analysis as well as modules for clustering multiple conformations and evaluation of order parameters for folding and aggregation. The program was developed with special emphasis on user-friendliness, effortless addition of new modules and efficient handling of large sets of trajectories.

Estimation of folding probabilities and phi values from molecular dynamics simulations of reversible Peptide folding.

Molecular dynamics simulations with an implicit model of the solvent have allowed to investigate the reversible folding of structured peptides. For a 20-residue antiparallel beta-sheet peptide, the simulation results have revealed multiple folding pathways. Moreover, the conformational heterogeneity of the denatured state has been shown to originate from high enthalpy, high entropy basins with fluctuating non-native secondary structure, as well as low enthalpy, low entropy traps.