Dimensionality of Carbon Nanomaterials Determines the Binding and Dynamics of Amyloidogenic Peptides: Multiscale Theoretical Simulations

Experimental studies have demonstrated that nanoparticles can affect the rate of protein self-assembly, possibly interfering with the development of protein misfolding diseases such as Alzheimer''s, Parkinson''s and prion disease caused by aggregation and fibril formation of amyloid-prone proteins. We employ classical molecular dynamics simulations and large-scale density functional theory calculations to investigate the effects of nanomaterials on the structure, dynamics and binding of an amyloidogenic peptide apoC-II(60-70). We show that the binding affinity of this peptide to carbonaceous nanomaterials such as C60, nanotubes and graphene decreases with increasing nanoparticle curvature. Strong binding is facilitated by the large contact area available for π-stacking between the aromatic residues of the peptide and the extended surfaces of graphene and the nanotube. The highly curved fullerene surface exhibits reduced efficiency for π-stacking but promotes increased peptide dynamics. We postulate that the increase in conformational dynamics of the amyloid peptide can be unfavorable for the formation of fibril competent structures. In contrast, extended fibril forming peptide conformations are promoted by the nanotube and graphene surfaces which can provide a template for fibril-growth.     

Multiple Time Step Brownian Dynamics for Long Time Simulation of Biomolecules

We report a multiple time step algorithm applied to an atomistic Brownian dynamics simulation for simulating the long time scale dynamics of biomolecules. The algorithm was based on the original multiple time step method; a short time step was used to keep faster motions in local equilibrium. When applied to a 28-mer # # ! folded peptide, the simulation gave stable trajectories and the computation time was reduced by a factor of 160 compared to a conventional molecular dynamics simulation using explicit water molecules. We applied it for the folding simulation of a 13-mer ! -helical peptide, giving a successful folding simulation. These results indicate that the Brownian dynamics with the multiple time step algorithm is useful for studies of biomolecular motions by long time simulation.     

Alamethicin in bicelles: Orientation,aggregation, and bilayer modification as a function of peptide concentration

Alamethicin is a 19-amino-acid residue hydrophobic peptide of the peptaibol family that has been the object of numerous studies for its ability to produce voltage-dependent ion channels in membranes. In this work, for the first time electron paramagnetic resonance spectroscopy was applied to study the interaction of alamethicin with oriented bicelles. We highlighted the effects of increasing peptide concentrations on both the peptide and the membrane in identical conditions, by adopting a twofold spin labeling approach, placing a nitroxide moiety either on the peptide or on the phospholipids. The employment of bicelles affords additional spectral resolution, thanks to the formation of a macroscopically oriented phase that allows to gain information on alamethicin orientation and dynamics. Moreover, the high viscosity of the bicellar solution permits the investigation of the peptide aggregation properties at physiological temperature. We observed that, at 35 °C, alamethicin adopts a transmembrane orientation with the peptide axis forming an average angle of 25° with respect to the bilayer normal. Moreover, alamethicin maintains its dynamics and helical tilt constant at all concentrations studied. On the other hand, by increasing the peptide concentration, the bilayer experiences an exponential decrease of the order parameter, but does not undergo micellization, even at the highest peptide to lipid ratio studied (1:20). Finally, the aggregation of the peptide at physiological temperature shows that the peptide is monomeric at peptide to lipid ratios lower than 1:50, then it aggregates with a rather broad distribution in the number of peptides (from 6 to 8) per oligomer.     

The Carboxy Terminus of the Ligand Peptide Determines the Stability of the MHC Class I Molecule H-2Kb: A Combined Molecular Dynamics and Experimental Study

Major histocompatibility complex (MHC) class I molecules (proteins) bind peptides of eight to ten amino acids to present them at the cell surface to cytotoxic T cells. The class I binding groove binds the peptide via hydrogen bonds with the peptide termini and via diverse interactions with the anchor residue side chains of the peptide. To elucidate which of these interactions is most important for the thermodynamic and kinetic stability of the peptide-bound state, we have combined molecular dynamics simulations and experimental approaches in an investigation of the conformational dynamics and binding parameters of a murine class I molecule (H-2K^b) with optimal and truncated natural peptide epitopes. We show that the F pocket region dominates the conformational and thermodynamic properties of the binding groove, and that therefore the binding of the C terminus of the peptide to the F pocket region plays a crucial role in bringing about the peptide-bound state of MHC class I.     

Role of hydrophobic interactions and salt-bridges in β-hairpin folding

β-Hairpins are the simplest form of β-sheets which, due to the presence of long-range interactions, can be considered as tertiary structures. Molecular dynamics simulation is a powerful tool that can unravel whole pathways of protein folding/unfolding at atomic resolution. We have performed several molecular dynamics simulations, to a total of over 250 ns, of a β-hairpin peptide in water using GROMACS. We show that hydrophobic interactions are necessary for initiating the folding of the peptide. Once formed, the peptide is stabilized by hydrogen bonds and disruption of hydrophobic interactions in the folded peptide does not denature the structure. In the absence of hydrophobic interactions, the peptide fails to fold. However, the introduction of a salt-bridge compensates for the loss of hydrophobic interactions to a certain extent. Figure Model of b-hairpin folding: Folding is initiated by hydrophobic interactions (Brown circles). The folded structure, once formed, is stabilized by hydrogen bonds (red lines) and is unaffected by loss of hydrophobic contacts     

Reversible Effects of Peptide Concentration and Lipid Composition on H-Ras Lipid Anchor Clustering

Dynamic clusters of lipid-anchored Ras proteins are important for high-fidelity signal transduction in cells. The average size of Ras nanoclusters was reported to be independent of protein expression levels, and cholesterol depletion is commonly used to test the raft-preference of nanoclusters. However, whether protein concentration and membrane domain stability affect Ras clustering in a reversible manner is not well understood. We used coarse-grained molecular dynamics simulations to examine the reversibility of the effects of peptide and cholesterol concentrations as well as a lipid domain-perturbing nanoparticle (C₆₀) on the dynamics and stability of H-Ras lipid-anchor nanoclusters. By comparing results from these simulations with previous observations from the literature, we show that effects of peptide/cholesterol concentrations on the dynamics and stability of H-Ras peptide nanoclusters are reversible. Our results also suggest a correlation between the stabilities of lipid domains and Ras nanoclusters, which is supported by our finding that C₆₀ penetrates into the liquid-disordered domain of the bilayer, destabilizing lipid domains and thereby the stability of the nanoclusters.     

Differential peptide dynamics is linked to major histocompatibility complex polymorphism

Peptide presentation by major histocompatibility complex (MHC) molecules is of central importance for immune responses, which are triggered through recognition of peptide-loaded MHC molecules (pMHC) by cellular ligands such as T-cell receptors (TCR). However, a unifying link between structural features of pMHC and cellular responses has not been established. Instead, pMHC/TCR binding studies suggest conformational and/or flexibility changes of the binding partners as a possible cause of differential T-cell stimulation, but information on real-time dynamics is lacking. We therefore probed the real-time dynamics of a MHC-bound nonapeptide (m9), by combining time-resolved fluorescence depolarization and molecular dynamics simulations. Here we show that the nanosecond dynamics of this peptide presented by two human MHC class I subtypes (HLA-B*2705 and HLA-B*2709) with differential autoimmune disease association varies dramatically, despite virtually identical crystal structures. The peptide dynamics is linked to the single, buried polymorphic residue 116 in the peptide binding groove. Pronounced peptide flexibility is seen only for the non-disease-associated subtype HLA-B*2709, suggesting an entropic control of peptide recognition. Thermodynamic data obtained for two additional peptides support this hypothesis.     

Cholesterol alters the inhibitory efficiency of peptide-based membrane fusion inhibitor

The membrane composition modulates membrane fusion by altering membrane physical properties and the structure, organization and dynamics of fusion proteins and peptides. The journey of developing peptide-based viral fusion inhibitors is often stalled by the change in lipid composition of viral and target membranes. This makes it important to study the role of membrane composition on the organization, dynamics and fusion inhibiting abilities of the peptide-based fusion inhibitors. Cholesterol, an important constituent of mammalian cell membrane, modulates bilayer properties in multiple ways and impart its effect on the membrane fusion. We have previously shown that TG-23 peptide derived from phagosomal coat protein, coronin 1, shows significant inhibition of fusion between membranes without cholesterol. In this work, we have studied the effect of the TG-23 peptide on the polyethylene glycol-mediated membrane fusion in presence of different concentrations of membrane cholesterol. Our results show that the inhibitory effect of TG-23 is being completely reversed in cholesterol containing membranes. We have evaluated the structure, organization, dynamics and depth of penetration of TG-23 in membranes having different lipid compositions and its effect on membrane properties. Our results demonstrate that cholesterol does not affect the secondary structure of the peptide, however, alters the depth of penetration of the peptide and modifies peptide organization and dynamics. The cholesterol dependent change in organization and dynamics of the peptide influences its efficacy in membrane fusion. Therefore, we envisage that the study of peptide organization and dynamics is extremely important to determine the effect of peptide on the membrane fusion.     

Nanodomain-induced chain folding in poly(gamma-benzyl-L-glutamate)-b-polyglycine diblock copolymers

We report on the self-assembly mechanism and dynamics in a series of poly(gamma-benzyl-l-glutamate)-b-poly(glycine) (PBLG-b-PGly) diblock copolymers within the composition range 0.67 < or = f(PBLG) < or = 0.97 and the temperature (T) range 303 < T < 433 K. Small- and wide-angle X-ray scattering, (13)C NMR, and differential scanning calorimetry are used for the structure investigation coupled with dielectric spectroscopy for both the peptide secondary structure and the associated dynamics. These techniques provide not only the nanophase morphology but also the type and persistence of peptide secondary structures. The thermodynamic confinement of the blocks within the nanodomains and the disparity in their packing efficiency results in multiple chain folding of the PGly secondary structure that effectively stabilize a lamellar morphology for high f(PBLG). Nanoscale confinement proves to be important in controlling the persistence length of secondary peptide motifs.     

Alzheimer's disease amyloid-β peptide analogue alters the ps-dynamics of phospholipid membranes

We have investigated the influence of the neurotoxic Alzheimer's disease peptide amyloid-β (25-35) on the dynamics of phospholipid membranes by means of quasi-elastic neutron scattering in the picosecond time-scale. Samples of pure phospholipids (DMPC/DMPS) and samples with amyloid-β (25-35) peptide included have been compared. With two different orientations of the samples the directional dependence of the dynamics was probed. The sample temperature was varied between 290 K and 320 K to cover both the gel phase and the liquid-crystalline phase of the lipid membranes. The model for describing the dynamics combines a long-range translational diffusion of the lipid molecules and a spatially restricted diffusive motion. Amyloid-β (25-35) peptide affects significantly the ps-dynamics of oriented lipid membranes in different ways. It accelerates the lateral diffusion especially in the liquid-crystalline phase. This is very important for all kinds of protein-protein interactions which are enabled and strongly influenced by the lateral diffusion such as signal and energy transducing cascades. Amyloid-β (25-35) peptide also increases the local lipid mobility as probed by variations of the vibrational motions with a larger effect in the out-of-plane direction. Thus, the insertion of amyloid-β (25-35) peptide changes not only the structure of phospholipid membranes as previously demonstrated by us employing neutron diffraction (disordering effect on the mosaicity of the lipid bilayer system) but also the dynamics inside the membranes. The amyloid-β (25-35) peptide induced membrane alteration even at only 3 mol% might be involved in the pathology of Alzheimer's disease as well as be a clue in early diagnosis and therapy.     

CONAN: A Tool to Decode Dynamical Information from Molecular Interaction Maps

The analysis of contacts is a powerful tool to understand biomolecular function in a series of contexts, from the investigation of dynamical behavior at equilibrium to the study of nonequilibrium dynamics in which the system moves between multiple states. We thus propose a tool called CONtact ANalysis (CONAN) that, from molecular dynamics (MD) trajectories, analyzes interresidue contacts, creates videos of time-resolved contact maps, and performs correlation, principal component, and cluster analysis, revealing how specific contacts relate to functionally relevant states sampled by MD. We present how CONAN can identify features describing the dynamics of ubiquitin both at equilibrium and during mechanical unfolding. Additionally, we show the analysis of MD trajectories of an α-synuclein mutant peptide that undergoes an α-β conformational transition that can be easily monitored using CONAN, which identifies the multiple states that the peptide explores along its conformational dynamics. The high versatility and ease of use of the software make CONAN a tool that can significantly facilitate the understanding of the complex dynamical behavior of proteins or other biomolecules. CONAN and its documentation are freely available for download on GitHub.     

Solution Structures,Dynamics, and Ice Growth Inhibitory Activity of Peptide Fragments Derived from an Antarctic Yeast Protein

Exotic functions of antifreeze proteins (AFP) and antifreeze glycopeptides (AFGP) have recently been attracted with much interest to develop them as commercial products. AFPs and AFGPs inhibit ice crystal growth by lowering the water freezing point without changing the water melting point. Our group isolated the Antarctic yeast Glaciozyma antarctica that expresses antifreeze protein to assist it in its survival mechanism at sub-zero temperatures. The protein is unique and novel, indicated by its low sequence homology compared to those of other AFPs. We explore the structure-function relationship of G. antarctica AFP using various approaches ranging from protein structure prediction, peptide design and antifreeze activity assays, nuclear magnetic resonance (NMR) studies and molecular dynamics simulation. The predicted secondary structure of G. antarctica AFP shows several α-helices, assumed to be responsible for its antifreeze activity. We designed several peptide fragments derived from the amino acid sequences of α-helical regions of the parent AFP and they also showed substantial antifreeze activities, below that of the original AFP. The relationship between peptide structure and activity was explored by NMR spectroscopy and molecular dynamics simulation. NMR results show that the antifreeze activity of the peptides correlates with their helicity and geometrical straightforwardness. Furthermore, molecular dynamics simulation also suggests that the activity of the designed peptides can be explained in terms of the structural rigidity/flexibility, i.e., the most active peptide demonstrates higher structural stability, lower flexibility than that of the other peptides with lower activities, and of lower rigidity. This report represents the first detailed report of downsizing a yeast AFP into its peptide fragments with measurable antifreeze activities.     

Position-dependent local motions in spin-labeled analogues of a short alpha-helical peptide determined by electron spin resonance.

We have used electron spin resonance and circular dichroism to examine and compare the dynamics in two analogues of the Ala-based 3K(I) peptide [Marqusee, S., Robbins, V.H., & Baldwin, R. L. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5286-5290], labeled at positions 4 and 8, throughout the alpha-helix----coil transition. In the middle of the thermal unfolding transition, our results demonstrate that the local mobility near the N-terminus is greater than at the center of the peptide. This provides evidence, from the perspective of dynamics, that the ends of Ala-based alpha-helices are frayed. We further find that the position dependence of the mobility for the thermally unfolded state differs from that of the denaturant unfolded state. Only the latter state exhibits the local dynamics expected for a genuine random coil.     

Motions of the SecA protein motor bound to signal peptide: Insights from molecular dynamics simulations

SecA is an essential part of the Sec pathway for protein secretion in bacteria. In this pathway, SecA interacts with the N-terminal fragment of the secretory protein – the signal peptide, and couples binding and hydrolysis of adenosine triphosphate with movement of the secretory protein across the SecY protein translocon. How interactions with the signal peptide alter the conformational dynamics and long-distance conformational couplings of SecA is a key open question that we address here with molecular dynamics techniques. Analyses of protein motions indicate that the signal peptide alters SecA dynamics not only at the site where this peptide binds, but also at a nucleotide-binding domain. Hydrogen bond clusters contribute to the long-distance propagation of changes in SecA dynamics.     

Computational study of solution behavior of magainin 2 monomers

Antimicrobial peptides (AMPs) play crucial role as mediators of the primary host defense against microbial invasion. They are considered a promising alternative to antibiotics for multidrug resistant bacterial strains. For complete understanding of the antimicrobial defense mechanism, a detailed knowledge of the dynamics of peptide-membrane interactions, including atomistic studies on AMPs geometry and both peptide and membrane structural changes during the whole process is a prerequisite. We aim at clarifying the conformation dynamics of small linear AMPs in solution as a first step of in silico protocol for establishing a correspondence between certain amino-acid sequence motifs, secondary-structure elements, conformational dynamics in solution and the intensity and mode of interaction with the bacterial membrane. To this end, we use molecular dynamics simulations augmented by well-tempered metadynamics to study the free-energy landscape of two AMPs with close primary structure and different antibacterial activity – the native magainin 2 (MG2) and an analog (MG2m, with substitutions F5Y and F16W) in aqueous solution. We observe that upon solvation, the initial α-helical structures change differently. The native form remains structured, with three shorter α-helical motifs, connected by random coils, while the synthetic analog tends predominantly to a disordered conformation. Our results indicate the importance of the side-chains at positions 5 and 16 for maintaining the solvated peptide conformation. They also provide a modeling background for recent experimental observations, relating the higher α-helical content in solution (peptide pre-folding) in the case of small linear AMPs to a lower antibacterial activity.     

A Comparative Approach Linking Molecular Dynamics of Altered Peptide Ligands and MHC with In Vivo Immune Responses

Background

The recognition of peptide in the context of MHC by T lymphocytes is a critical step in the initiation of an adaptive immune response. However, the molecular nature of the interaction between peptide and MHC and how it influences T cell responsiveness is not fully understood.

Results

We analyzed the immunological consequences of the interaction of MHC class II (I-A^u) restricted 11-mer peptides of myelin basic protein with amino acid substitutions at position 4. These mutant peptides differ in MHC binding affinity, CD4⁺ T cell priming, and alter the severity of peptide-induced experimental allergic encephalomyelitis. Using molecular dynamics, a computational method of quantifying intrinsic movements of proteins at high resolution, we investigated conformational changes in MHC upon peptide binding. We found that irrespective of peptide binding affinity, MHC deformation appears to influence costimulation, which then leads to effective T cell priming and disease induction. Although this study compares in vivo and molecular dynamics results for three altered peptide ligands, further investigation with similar complexes is essential to determine whether spatial rearrangement of peptide-MHC and costimulatory complexes is an additional level of T cell regulation.     

Membrane interactions and dynamics of a 21-mer cytotoxic peptide: A solid-state NMR study

We have investigated the membrane interactions and dynamics of a 21-mer cytotoxic model peptide that acts as an ion channel by solid-state NMR spectroscopy. To shed light on its mechanism of membrane perturbation, ³¹P and ²H NMR experiments were performed on 21-mer peptide-containing bicelles. ³¹P NMR results indicate that the 21-mer peptide stabilizes the bicelle structure and orientation in the magnetic field and perturbs the lipid polar head group conformation. On the other hand, ²H NMR spectra reveal that the 21-mer peptide orders the lipid acyl chains upon binding. ¹⁵N NMR experiments performed in DMPC bilayers stacked between glass plates also reveal that the 21-mer peptide remains at the bilayer surface. ¹⁵N NMR experiments in perpendicular DMPC bicelles indicate that the 21-mer peptide does not show a circular orientational distribution in the bicelle planar region. Finally, ¹³C NMR experiments were used to study the 21-mer peptide dynamics in DMPC multilamellar vesicles. By analyzing the ¹³CO spinning sidebands, the results show that the 21-mer peptide is immobilized upon membrane binding. In light of these results, we propose a model of membrane interaction for the 21-mer peptide where it lies at the bilayer surface and perturbs the lipid head group conformation.     

Lateral dynamics of proteins with polybasic domain on anionic membranes: a dynamic Monte-Carlo study

Positively charged polybasic domains are essential for recruiting multiple signaling proteins, such as Ras GTPases and Src kinase, to the negatively charged cellular membranes. Much less, however, is known about the influence of electrostatic interactions on the lateral dynamics of these proteins. We developed a dynamic Monte-Carlo automaton that faithfully simulates lateral diffusion of the adsorbed positively charged oligopeptides as well as the dynamics of mono- (phosphatidylserine) and polyvalent (PIP₂) anionic lipids within the bilayer. In agreement with earlier results, our simulations reveal lipid demixing that leads to the formation of a lipid shell associated with the peptide. The computed association times and average numbers of bound lipids demonstrate that tetravalent PIP₂ interacts with the peptide much more strongly than monovalent lipid. On the spatially homogeneous membrane, the lipid shell affects the behavior of the peptide only by weakly reducing its lateral mobility. However, spatially heterogeneous distributions of monovalent lipids are found to produce peptide drift, the velocity of which is determined by the total charge of the peptide-lipid complex. We hypothesize that this predicted phenomenon may affect the spatial distribution of proteins with polybasic domains in the context of cell-signaling events that alter the local density of monovalent anionic lipids.