Abstract: Community network analysis derived from molecular dynamics simulations is used to identify and compare the signaling pathways in a bacterial glutamyl-tRNA synthetase (GluRS):tRNA(Glu) complex as well as the tRNA migration complex, GluRS:Glu-tRNA(Glu):GTP:EF-Tu. A dynamic contact map defines the edges connecting nodes (amino acids and nucleotides) in the physical network whose overall topology is presented as a network of communities, local substructures that are highly intraconnected, but loosely interconnected. While nodes within a single community can communicate through many alternate pathways, the communication between monomers in different communities has to take place through a smaller number of critical edges or interactions. There are a large number of suboptimal paths that can also be used for communication between the identity elements on the tRNAs and the catalytic site in the GluRS:tRNA complex. Residues and nucleotides in the majority of connections for intercommunity signal transmission are evolutionarily conserved and are predicted to be important for allosteric signaling. The same monomers are also found in a majority of the suboptimal paths. Modifying these residues or nucleotides has a large effect on the communication pathways in the protein:RNA complex consistent with kinetic data. It has been reported that the presence of EF-Tu stimulates the dissociation of charged aa-tRNA from several class I aaRSs. Using conformations observed in molecular dynamics trajectories of the isolated systems, a tRNA migration complex was assembled free of steric clashes. This complex was used to study the migration of tRNA from GluRS to EF-Tu. While the full migration (~50 nm) has not yet been observed, network analysis reveals the dissolution of GluRS:tRNA communities as well as formation of EF-Tu:tRNA communities as the tRNA migrates from one protein to the other.

Abstract: During the elongation cycle, tRNA and mRNA undergo coupled translocation through the ribosome catalyzed by elongation factor G (EF-G). Cryo-EM reconstructions of certain EF-G-containing complexes led to the proposal that the mechanism of translocation involves rotational movement between the two ribosomal subunits. Using single-molecule FRET, we observe that pretranslocation ribosomes undergo spontaneous intersubunit rotational movement in the absence of EF-G, fluctuating between two conformations corresponding to the classical and hybrid states of the translocational cycle. In contrast, posttranslocation ribosomes are fixed predominantly in the classical, nonrotated state. Movement of the acceptor stem of deacylated tRNA into the 50S E site and EF-G binding to the ribosome both contribute to stabilization of the rotated, hybrid state. Furthermore, the acylation state of P site tRNA has a dramatic effect on the frequency of intersubunit rotation. Our results provi de direct evidence that the intersubunit rotation that underlies ribosomal translocation is thermally driven. In a separate series of experiments, we observed L1 stalk dynamics using 70S ribosomes with the donor and acceptor dyes both on the 50S subunit. In pretranslocation ribosomes, the L1 stalk fluctuates between just two distinct states suggesting that L1 stalk movement and ratcheting are correlated. However, several translational intermediate states are not correlated with ratcheting suggesting that L1 stalk movement can be decoupled from ribosomal ratcheting during translational elongation.

Abstract: In the photosynthetic purple bacterium Rhodobacter (Rba.) sphaeroides, light is absorbed by membrane-bound light-harvesting (LH) proteins LH1 and LH2. LH1 directly surrounds the reaction center (RC) and, together with PufX, forms a dimeric (RC-LH1-PufX)2 protein complex. In LH2-deficient Rba. sphaeroides mutants, RC-LH1-PufX dimers aggregate into tubular vesicles with a radius of ~250-550A, making RC-LH1-PufX one of the few integral membrane proteins known to actively induce membrane curvature. Recently, a three-dimensional electron microscopy density map showed that the Rba. sphaeroides RC-LH1-PufX dimer exhibits a prominent bend at its dimerizing interface. To investigate the curvature properties of this highly bent protein, we employed molecular dynamics simulations to fit an all-atom structural model of the RC-LH1-PufX dimer within the electron microscopy density map. The simulations reveal how the dimer produces a membrane with high local curvature, even t hough the location of PufX cannot yet be determined uniquely. The resulting membrane curvature agrees well with the size of RC-LH1-PufX tubular vesicles, and demonstrates how the local curvature properties of the RC-LH1-PufX dimer propagate to form the observed long-range organization of the Rba. sphaeroides tubular vesicles.

Abstract: Upon infection of E. coli by bacteriophage lambda, a decision is made between the lytic pathway, leading to cell death and the release of new phages; and the lysogenic pathway, in which the phage genome integrates into the bacterial chromosome. In this study, we use live fluorescence microscopy to follow lambda infection and the resulting cell fate at the level of individual cells and phages, with the aim of elucidating the different parameters affecting decision-making. In addition, we follow phage trajectories in space to learn how a single phage finds its target on the host cell.

Abstract: With the recent discovery of several crystal structures of complete ABC transporters, an alternating access model for substrate transport has been hypothesized, in which the transporter is open to the cytoplasm in the resting state and only accessible extracellularly in its ATP-bound, intermediate state. To test the hypothesized transport mechanism, we use molecular dynamics simulations to investigate the conformational changes and detailed interactions between structural components of ABC transporters in a membrane environment. Starting from the crystal structure of an intact maltose transporter which is trapped in the intermediate state, 50 ns or longer simulations are performed on the complete transporter, as well as on the transmembrane domains (TMDs) in the presence or absence of other components, and the conformational coupling of different domains is analyzed. We find that in the presence of nucleotide binding domains (NBDs) and the absence of nucleotides, the TMDs tend to open the cytoplasmic end, consistent with the prevailing transport mechanism. However, the cytoplasmic opening is not observed when the NBDs are absent, suggesting that the cytoplasmic-open state is dictated by the separation of the NBDs, and not as a result of the natural tendency of the TMDs to stay open. Furthermore, the results show that the opening of NBDs is propagated to TMDs through the mechanical engagement of the two helices at the EAA loop of the TMDs, which requires the formation of a 3-helix bundle together with the helix next to the Q-loop at the NBD helical subdomain. In the absence of NBDs the two coupling helices are completely decoupled from the rest of the TMDs, undergoing large fluctuations relative to the rigid TMD structures and show no conformational correlation to the other two EAA helices.

Abstract: Magic-angle spinning solid-state NMR (MAS-SSNMR) has emerged as a premier technique in structural biology for obtaining atomic resolution information on large proteins and protein complexes. Many such systems, including membrane proteins and insoluble aggregates, pose extreme challenges to solution NMR and X-ray crystallography, placing SSNMR in a unique position to provide important structural details. Alpha-synuclein (AS) is a human protein that forms fibrils and constitutes the primary protein component of Lewy Bodies, the pathological hallmark of Parkinson's disease. AS fibrils are insoluble and do not diffract; therefore SSNMR is the only route to atomic resolution structural information. As a route to the structure of AS fibrils, an array of NMR experiments has been developed on the small protein GB1. The spectra from these experiments enable the determination of inter-atomic distances, backbone and side-chain torsion angles and intermolecular contacts. These NMR data are incorporated into simulated annealing molecular dynamics calculations to solve the monomer and multimeric nanocrystalline structure of GB1. All of these techniques are now being applied to AS fibrils with the goal of solving the atomic resolution structure of AS fibrils. This structure will be vital in understanding the effects of AS fibrils on neurons and could provide insight into potential therapies and drugs for Parkinson's disease.