The all-atom additive CHARMM36 protein force field is widely used in molecular modeling and simulations. We present its refinement, CHARMM36m (http://mackerell.umaryland.edu/charmm_ff.shtml), with improved accuracy in generating polypeptide backbone conformational ensembles for intrinsically disordered peptides and proteins.

Membrane traffic in eukaryotic cells involves transport of vesicles that bud from a donor compartment and fuse with an acceptor compartment. Common principles of budding and fusion have emerged, and many of the proteins involved in these events are now known. However, a detailed picture of an entire trafficking organelle is not yet available. Using synaptic vesicles as a model, we have now determined the protein and lipid composition; measured vesicle size, density, and mass; calculated the average protein and lipid mass per vesicle; and determined the copy number of more than a dozen major constituents. A model has been constructed that integrates all quantitative data and includes structural models of abundant proteins. Synaptic vesicles are dominated by proteins, possess a surprising diversity of trafficking proteins, and, with the exception of the V-ATPase that is present in only one to two copies, contain numerous copies of proteins essential for membrane traffic and neurotransmitter uptake.

Protein dynamics are essential for protein function, and yet it has been challenging to access the underlying atomic motions in solution on nanosecond-to-microsecond time scales. We present a structural ensemble of ubiquitin, refined against residual dipolar couplings (RDCs), comprising solution dynamics up to microseconds. The ensemble covers the complete structural heterogeneity observed in 46 ubiquitin crystal structures, most of which are complexes with other proteins. Conformational selection, rather than induced-fit motion, thus suffices to explain the molecular recognition dynamics of ubiquitin. Marked correlations are seen between the flexibility of the ensemble and contacts formed in ubiquitin complexes. A large part of the solution dynamics is concentrated in one concerted mode, which accounts for most of ubiquitin's molecular recognition heterogeneity and ensures a low entropic complex formation cost.

The force required to rupture the streptavidin-biotin complex was calculated here by computer simulations. The computed force agrees well with that obtained by recent single molecule atomic force microscope experiments. These simulations suggest a detailed multiple-pathway rupture mechanism involving five major unbinding steps. Binding forces and specificity are attributed to a hydrogen bond network between the biotin ligand and residues within the binding pocket of streptavidin. During rupture, additional water bridges substantially enhance the stability of the complex and even dominate the binding interactions. In contrast, steric restraints do not appear to contribute to the binding forces, although conformational motions were observed.

Electrostatic interactions govern structural and dynamical properties of membranes and can vary considerably with the composition of the aqueous buffer. We studied the influence of sodium chloride on a pure POPC lipid bilayer by fluorescence correlation spectroscopy experiments and molecular dynamics simulations. Increasing sodium chloride concentration was found to decrease the self-diffusion of POPC lipids within the bilayer. Self-diffusion coefficients calculated from the 100 ns simulations agree with those measured on a millisecond timescale, suggesting that most of the relaxation processes relevant for lipid diffusion are faster than the simulation timescale. As the dominant effect, the molecular dynamics simulations revealed a tight binding of sodium ions to the carbonyl oxygens of on average three lipids leading to larger complexes with reduced mobility. Additionally, the bilayer thickens by approximately 2 A, which increases the order parameter of the fatty acyl chains. Sodium binding alters the electrostatic potential, which is largely compensated by a changed polarization of the aqueous medium and a lipid dipole reorientation.

We present a method to predict complex structural (conformational) transitions in irregular or disordered macromolecular systems, such as proteins or glasses, at the atomic level. Our method aims at rare events, which currently cannot be predicted with traditional molecular dynamics (MD) simulations, since these currently are limited to time scales shorter than a few nanoseconds. Given an initial conformation of the system, our method identi6es one or more product states, which may be separated from the initial state by free energy barriers that are large on the scale of thermal energy. It also provides an approximate reaction path, which can be used to determine barrier heights or reaction rates with the usual techniques. The method employs an arti6cial potential that destabilizes the initial conformation and, thereby, lowers free energy barriers of structural transitions. As a result, transitions are accelerated and may be observed in MD simulations. An analytical estimate for the acceleration factor is given. The method is applied to two test systems, an argon microcluster and a simpli6ed protein model. By these studies we demonstrated that our method is capable of shortening mean transition times from 0.5 ps (argon cluster) and 1.4 ns (protein model) to a few picoseconds. These results suggest that our method is particularly well suited to study biochemically relevant conformational motions in proteins at a microsecond time scale.

Neuronal exocytosis is catalyzed by the SNARE protein syntaxin-1A1. Syntaxin-1A is clustered in the plasma membrane at sites where synaptic vesicles undergo exocytosis2,3. However, how syntaxin-1A is sequestered is unknown. Here, we show that syntaxin clustering is mediated by electrostatic interactions with the strongly anionic lipid phosphatidylinositol-4,5-bisphosphate (PIP2). We found with super-resolution STED microscopy on the plasma membrane of PC12 cells that PIP2 is the dominant inner-leaflet lipid in ~73 nm-sized microdomains. This high accumulation of PIP2 was required for syntaxin-1A sequestering, as destruction of PIP2 by the phosphatase synaptojanin-1 reduced syntaxin-1A clustering. Furthermore, co-reconstitution of PIP2 and the C-terminal part of syntaxin-1A in artificial giant unilamellar vesicles resulted in segregation of PIP2 and syntaxin-1A into distinct domains even when cholesterol was absent. Our results demonstrate that electrostatic protein-lipid interactions can result in the formation of microdomains independent of cholesterol or lipid phases.

Most plasmalemmal proteins organize in submicrometer-sized clusters whose architecture and dynamics are still enigmatic. With syntaxin 1 as an example, we applied a combination of far-field optical nanoscopy, biochemistry, fluorescence recovery after photobleaching (FRAP) analysis, and simulations to show that clustering can be explained by self-organization based on simple physical principles. On average, the syntaxin clusters exhibit a diameter of 50 to 60 nanometers and contain 75 densely crowded syntaxins that dynamically exchange with freely diffusing molecules. Self-association depends on weak homophilic protein-protein interactions. Simulations suggest that clustering immobilizes and conformationally constrains the molecules. Moreover, a balance between self-association and crowding-induced steric repulsions is sufficient to explain both the size and dynamics of syntaxin clusters and likely of many oligomerizing membrane proteins that form supramolecular structures.