Analyses of protein cores reveal fundamental differences between solution and crystal structures.

There have been several studies suggesting that protein structures solved by NMR spectroscopy and X-ray crystallography show significant differences. To understand the origin of these differences, we assembled a database of high-quality protein structures solved by both methods. We also find significant differences between NMR and crystal structures-in the root-mean-square deviations of the C α atomic positions, identities of core amino acids, backbone, and side-chain dihedral angles, and packing fraction of core residues. In contrast to prior studies, we identify the physical basis for these differences by modeling protein cores as jammed packings of amino acid-shaped particles. We find that we can tune the jammed packing fraction by varying the degree of thermalization used to generate the packings. For an athermal protocol, we find that the average jammed packing fraction is identical to that observed in the cores of protein structures solved by X-ray crystallography. In contrast, highly thermalized packing-generation protocols yield jammed packing fractions that are even higher than those observed in NMR structures. These results indicate that thermalized systems can pack more densely than athermal systems, which suggests a physical basis for the structural differences between protein structures solved by NMR and X-ray crystallography.

The Mitochondrial Peptide Humanin Targets but Does Not Denature Amyloid Oligomers in Type II Diabetes.

Mitochondrially derived peptides (MDPs) such as humanin (HN) have shown a remarkable ability to modulate neurological amyloids and apoptosis-associated proteins in cells and animal models. Recently, we found that humanin-like peptides also inhibit amyloid formation outside of neural environments in islet amyloid polypeptide (IAPP) fibrils and plaques, which are hallmarks of Type II diabetes. However, the biochemical basis for regulating amyloids through endogenous MDPs remains elusive. One hypothesis is that MDPs stabilize intermediate amyloid oligomers and discourage the formation of insoluble fibrils. To test this hypothesis, we carried out simulations and experiments to extract the dominant interactions between the S14G-HN mutant (HNG) and a diverse set of IAPP structures. Replica-exchange molecular dynamics suggests that MDPs cap the growth of amyloid oligomers. Simulations also indicate that HNG-IAPP heterodimers are 10 times more stable than IAPP homodimers, which explains the substoichiometric ability of HNG to inhibit amyloid growth. Despite this strong attraction, HNG does not denature IAPP. Instead, HNG binds IAPP near the disordered NFGAIL motif, wedging itself between amyloidogenic fragments. Shielding of NFGAIL-flanking fragments reduces the formation of parallel IAPP ß-sheets and subsequent nucleation of mature amyloid fibrils. From ThT spectroscopy and electron microscopy, we found that HNG does not deconstruct mature IAPP fibrils and oligomers, consistent with the simulations and our proposed hypothesis. Taken together, this work provides new mechanistic insight into how endogenous MDPs regulate pathological amyloid growth at the molecular level and in highly substoichiometric quantities, which can be exploited through peptidomimetics in diabetes or Alzheimer's disease.

Surface force measurements and simulations of mussel-derived peptide adhesives on wet organic surfaces.

Translating sticky biological molecules-such as mussel foot proteins (MFPs)-into synthetic, cost-effective underwater adhesives with adjustable nano- and macroscale characteristics requires an intimate understanding of the glue's molecular interactions. To help facilitate the next generation of aqueous adhesives, we performed a combination of surface forces apparatus (SFA) measurements and replica-exchange molecular dynamics (REMD) simulations on a synthetic, easy to prepare, Dopa-containing peptide (MFP-3s peptide), which adheres to organic surfaces just as effectively as its wild-type protein analog. Experiments and simulations both show significant differences in peptide adsorption on CH3-terminated (hydrophobic) and OH-terminated (hydrophilic) self-assembled monolayers (SAMs), where adsorption is strongest on hydrophobic SAMs because of orientationally specific interactions with Dopa. Additional umbrella-sampling simulations yield free-energy profiles that quantitatively agree with SFA measurements and are used to extract the adhesive properties of individual amino acids within the context of MFP-3s peptide adhesion, revealing a delicate balance between van der Waals, hydrophobic, and electrostatic forces.

Regulation and aggregation of intrinsically disordered peptides.

Intrinsically disordered proteins (IDPs) are a unique class of proteins that have no stable native structure, a feature that allows them to adopt a wide variety of extended and compact conformations that facilitate a large number of vital physiological functions. One of the most well-known IDPs is the microtubule-associated tau protein, which regulates microtubule growth in the nervous system. However, dysfunctions in tau can lead to tau oligomerization, fibril formation, and neurodegenerative disease, including Alzheimer's disease. Using a combination of simulations and experiments, we explore the role of osmolytes in regulating the conformation and aggregation propensities of the R2/wt peptide, a fragment of tau containing the aggregating paired helical filament (PHF6*). We show that the osmolytes urea and trimethylamine N-oxide (TMAO) shift the population of IDP monomer structures, but that no new conformational ensembles emerge. Although urea halts aggregation, TMAO promotes the formation of compact oligomers (including helical oligomers) through a newly proposed mechanism of redistribution of water around the perimeter of the peptide. We put forth a "superposition of ensembles" hypothesis to rationalize the mechanism by which IDP structure and aggregation is regulated in the cell.

Molecularly Smooth Self-Assembled Monolayer for High-Mobility Organic Field-Effect Transistors.

Despite the need for molecularly smooth self-assembled monolayers (SAMs) on silicon dioxide surfaces (the most common dielectric surface), current techniques are limited to nonideal silane grafting. Here, we show unique bioinspired zwitterionic molecules forming a molecularly smooth and uniformly thin SAM in "water" in <1 min on various dielectric surfaces, which enables a dip-coating process that is essential for organic electronics to become reality. This monomolecular layer leads to high mobility of organic field-effect transistors (OFETs) based on various organic semiconductors and source/drain electrodes. A combination of experimental and computational techniques confirms strong adsorption (Wad > 20 mJ m-2), uniform thickness (∼0.5 or ∼1 nm) and orientation (all catechol head groups facing the oxide surface) of the "monomolecular" layers. This robust (strong adsorption), rapid, and green SAM represents a promising advancement toward the next generation of nanofabrication compared to the current nonuniform and inconsistent polysiloxane-based SAM involving toxic chemicals, long processing time (>10 h), or heat (>80 °C).

Phospholipid and Hydrocarbon Interactions with a Charged Electrode Interface.

Using a combination of molecular dynamics simulations and experiments we examined the interactions of alkanes and phospholipids at charged interfaces in order to understand how interfacial charge densities affect the association of these two representative molecules with electrodes. Consistent with theory and experiment, these model systems reveal interfacial associations mediated through a combination of Coulombic and van der Waals forces. van der Waals forces, in particular, mediate rapid binding of decane to neutral electrodes. No decane binding was observed at high surface charge densities because of interfacial water polarization, which screens hydrophobic attractions. The positively charged choline moiety of the phospholipid palmitoyloleoylphosphatidylcholine (POPC) is primarily responsible for POPC attraction by a moderately negatively charged electrode. The hydrocarbon tails of POPC interact with the hydrophobic electrode interface similarly to decane. Previously reported electrochemical results confirm these findings by demonstrating bipolar displacement currents from PC vesicles adhering to moderately negatively charged interfaces, originating from the choline interactions observed in simulations. At more negatively charged interfaces, choline-to-surface binding was stronger. In both simulations and experiments the maximal interaction of anionic PS occurs with a positively charged interface, provided that the electrostatic forces outweigh local Lennard-Jones interactions. Direct comparisons between the binding affinities measured in experiments and those obtained in simulations reveal previously unobserved atomic interactions that facilitate lipid vesicle adhesion to charged interfaces. Moreover, the implementation of a charged interface in molecular dynamics simulations provides an alternative method for the generation of large electric fields across phospholipid bilayers, especially for systems with periodic boundary conditions, and may be useful for simulations of membrane electropermeabilization.

Picosecond and Terahertz Perturbation of Interfacial Water and Electropermeabilization of Biological Membranes.

Non-thermal probing and stimulation with subnanosecond electric pulses and terahertz electromagnetic radiation may lead to new, minimally invasive diagnostic and therapeutic procedures and to methods for remote monitoring and analysis of biological systems, including plants, animals, and humans. To effectively engineer these still-emerging tools, we need an understanding of the biophysical mechanisms underlying the responses that have been reported to these novel stimuli. We show here that subnanosecond (≤500 ps) electric pulses induce action potentials in neurons and cause calcium transients in neuroblastoma-glioma hybrid cells, and we report complementary molecular dynamics simulations of phospholipid bilayers in electric fields in which membrane permeabilization occurs in less than 1 ns. Water dipoles in the interior of these model membranes respond in less than 1 ps to permeabilizing electric potentials by aligning in the direction of the field, and they re-orient at terahertz frequencies to field reversals. The mechanism for subnanosecond lipid electropore formation is similar to that observed on longer time scales-energy-minimizing intrusions of interfacial water into the membrane interior and subsequent reorganization of the bilayer into hydrophilic, conductive structures.

To What Extent Does Surface Hydrophobicity Dictate Peptide Folding and Stability near Surfaces?

Protein-surface interactions are ubiquitous in both the cellular setting and in modern bioengineering devices, but how such interactions impact protein stability is not well understood. We investigate the folding of the GB1 hairpin peptide in the presence of self-assembled monolayers and graphite like surfaces using replica exchange molecular dynamics simulations. By varying surface hydrophobicity, and decoupling direct protein-surface interactions from water-mediated interactions, we show that surface wettability plays a surprisingly minor role in dictating protein stability. For both the ß-hairpin GB1 and the helical miniprotein TrpCage, adsorption and stability is largely dictated by the nature of the direct chemical interactions between the protein and the surface. Independent of the surface hydrophobicity profile, strong protein-surface interactions destabilize the folded structure while weak interactions stabilize it.

Water influx and cell swelling after nanosecond electropermeabilization.

Pulsed electric fields are used to permeabilize cell membranes in biotechnology and the clinic. Although molecular and continuum models provide compelling representations of the mechanisms underlying this phenomenon, a clear structural link between the biomolecular transformations displayed in molecular dynamics (MD) simulations and the micro- and macroscale cellular responses observed in the laboratory has not been established. In this paper, plasma membrane electropermeabilization is characterized by exposing Jurkat T lymphoblasts to pulsed electric fields less than 10ns long (including single pulse exposures), and by monitoring the resulting osmotically driven cell swelling as a function of pulse number and pulse repetition rate. In this way, we reduce the complexity of the experimental system and lay a foundation for gauging the correspondence between measured and simulated values for water and ion transport through electropermeabilized membranes. We find that a single 10MV/m pulse of 5ns duration produces measurable swelling of Jurkat T lymphoblasts in growth medium, and we estimate from the swelling kinetics the ion and water flux that follows the electropermeabilization of the membrane. From these observations we set boundaries on the net conductance of the permeabilized membrane, and we show how this is consistent with model predictions for the conductance and areal density of nanoelectropulse-induced lipid nanopores.

Determination of biomembrane bending moduli in fully atomistic simulations.

The bilayer bending modulus (Kc) is one of the most important physical constants characterizing lipid membranes, but precisely measuring it is a challenge, both experimentally and computationally. Experimental measurements on chemically identical bilayers often differ depending upon the techniques employed, and robust simulation results have previously been limited to coarse-grained models (at varying levels of resolution). This Communication demonstrates the extraction of Kc from fully atomistic molecular dynamics simulations for three different single-component lipid bilayers (DPPC, DOPC, and DOPE). The results agree quantitatively with experiments that measure thermal shape fluctuations in giant unilamellar vesicles. Lipid tilt, twist, and compression moduli are also reported.