Application of classical molecular dynamics for evaluation of proton transfer mechanism on a protein

AbstractProton transfer reactions on surfaces are prevalent in biology, chemistry and physics. In the present study, we employed classical Molecular Dynamics simulations to search for the presence of transient configurations that enable proton transfer, or proton sharing, between adjacent carboxylate groups on the protein surface. The results demonstrate that, during random fluctuations of the residues on the surface, there are repeated situations in which nearby carboxylates either share a common proton through a hydrogen bond, or are connected by a few water molecules that form conducting networks. These networks do not extend out of the common Coulomb cage of the participating residues and the lifetimes of the bridged structures are sufficiently long to allow passage of a proton between the carboxylates. The detection of domains capable of supporting a rapid proton transfer on a protein supports the notion that clusters of carboxylates are the operative elements of proton collecting antennae, as in bacteriorhodopsin, cytochrome c oxidase or the photosynthetic reaction center

Imaging the assembly and disassembly kinetics of cis-SNARE complexes on native plasma membranes

AbstractMild sonication of eukaryotic cells produces native plasma membrane sheets that retain their docked organelles, cytoskeleton structures and cytoplasmic complexes. While the delicate organization of membranous protein complexes remains undisturbed, their inner plasmalemmel leaflet can be rapidly exposed to bathing solutions, enabling specific biochemical manipulations. Here, we apply this system to track membrane-biochemistry kinetics. We monitor soluble NSF-attachment protein receptor (SNARE) complex assembly and disassembly on the plasma membrane at high time resolution. The results suggest two-phase kinetics for the assembly process and dependence of the disassembly kinetics on both N-ethyl maleimide-sensitive factor (NSF) and soluble NSF-attachment protein (α-SNAP) concentrations

Super-resolution imaging reveals the internal architecture of nano-sized syntaxin clusters

Key synaptic proteins from the soluble SNARE (N-ethylmaleimide-sensitive factor attachment protein receptor) family, among many others, are organized at the plasma membrane of cells as clusters containing dozens to hundreds of protein copies. However, the exact membranal distribution of proteins into clusters or as single molecules, the organization of molecules inside the clusters, and the clustering mechanisms are unclear due to limitations of the imaging and analytical tools. Focusing on syntaxin 1 and SNAP-25, we implemented direct stochastic optical reconstruction microscopy together with quantitative clustering algorithms to demonstrate a novel approach to explore the distribution of clustered and nonclustered molecules at the membrane of PC12 cells with single-molecule precision. Direct stochastic optical reconstruction microscopy images reveal, for the first time, solitary syntaxin/SNAP-25 molecules and small clusters as well as larger clusters. The nonclustered syntaxin or SNAP-25 molecules are mostly concentrated in areas adjacent to their own clusters. In the clusters, the density of the molecules gradually decreases from the dense cluster core to the periphery. We further detected large clusters that contain several density gradients. This suggests that some of the clusters are formed by unification of several clusters that preserve their original organization or reorganize into a single unit. Although syntaxin and SNAP-25 share some common distributional features, their clusters differ markedly from each other. SNAP-25 clusters are significantly larger, more elliptical, and less dense. Finally, this study establishes methodological tools for the analysis of single-molecule-based super-resolution imaging data and paves the way for revealing new levels of membranal protein organization

The Dynamics of Ca2+ Ions within the Solvation Shell of Calbindin D9k

The encounter of a Ca2+ ion with a protein and its subsequent binding to specific binding sites is an intricate process that cannot be fully elucidated from experimental observations. We have applied Molecular Dynamics to study this process with atomistic details, using Calbindin D9k (CaB) as a model protein. The simulations show that in most of the time the Ca2+ ion spends within the Debye radius of CaB, it is being detained at the 1st and 2nd solvation shells. While being detained near the protein, the diffusion coefficient of the ion is significantly reduced. However, due to the relatively long period of detainment, the ion can scan an appreciable surface of the protein. The enhanced propagation of the ion on the surface has a functional role: significantly increasing the ability of the ion to scan the protein's surface before being dispersed to the bulk. The contribution of this mechanism to Ca2+ binding becomes significant at low ion concentrations, where the intervals between successive encounters with the protein are getting longer. The efficiency of the surface diffusion is affected by the distribution of charges on the protein's surface. Comparison of the Ca2+ binding dynamics in CaB and its E60D mutant reveals that in the wild type (WT) protein the carboxylate of E60 function as a preferred landing-site for the Ca2+ arriving from the bulk, followed by delivering it to the final binding site. Replacement of the glutamate by aspartate significantly reduced the ability to transfer Ca2+ ions from D60 to the final binding site, explaining the observed decrement in the affinity of the mutated protein to Ca2+

The Role of Small Intraprotein Cavities in the Catalytic Cycle of Bacteriorhodopsin

The last phase of the proton transfer cycle of bacteriorhodopsin calls for a passage of a proton from D38 to D96. This reaction utilizes a narrow shaft ∼10-Å long that connects the two carboxylates that cross through a very hydrophobic domain. As the shaft is too narrow to be permanently hydrated, there are two alternatives for the proton propagation into the channel. The proton may propagate through the shaft without solvation at the expense of a high electrostatic barrier; alternatively, the shaft will expand to accommodate some water molecules, thus lowering the Born energy for the insertion of the charge into the protein (B. Schätzler, N. A. Dencher, J. Tittor, D. Oesterhelt, S. Yaniv-Checover, E. Nachliel, and G. Gutman, 2003, Biophys. J. 84:671–686). A comparative study of nine published crystal-structures of bacteriorhodopsin identified, next to the shaft, microcavities in the protein whose position and surrounding atoms are common to the reported structures. Some of the cavities either shrink or expand during the photocycle. It is argued that the plasticity of the cavities provides a working space needed for the transient solvation of the shaft, thus reducing the activation energy necessary for the solvation of the shaft. This suggestion is corroborated by the recent observations of Klink et al. (B. U. Klink, R. Winter, M. Engelhard, and I. Chizhov, 2002, Biophys. J. 83:3490–3498) that the late phases of the photocycle (τ ≥ 1 ms) are strongly inhibited by external pressure

Gauging of the PhoE channel by a single freely diffusing proton.

In the present study we combined a continuum approximation with a detailed mapping of the electrostatic potential inside an ionic channel to define the most probable trajectory for proton propagation through the channel (propagation along a structure-supported trajectory (PSST)). The conversion of the three-dimensional diffusion space into propagation along a one-dimensional pathway permits reconstruction of an ion motion by a short calculation (a few seconds on a state-of-the-art workstation) rather than a laborious, time-consuming random walk simulations. The experimental system selected for testing the accuracy of this concept was the reversible dissociation of a proton from a single pyranine molecule (8-hydroxypyrene-1,2,3-trisulfonate) bound by electrostatic forces inside the PhoE ionic channel of the Escherichia coli outer membrane. The crystal structure coordinates were used for calculation of the intra-cavity electrostatic potential, and the reconstruction of the observed fluorescence decay curve was carried out using the dielectric constant of the intra-cavity space as an adjustable parameter. The fitting of past experimental observations (Shimoni, E., Y. Tsfadia, E. Nachliel, and M. Gutman. 1993. Biophys. J. 64:472-479) was carried out by a modified version of the Agmon geminate recombination program (Krissinel, E. B., and N. Agmon. 1996. J. Comp. Chem. 17:1085-1098), where the gradient of the electrostatic potential and the entropic terms were calculated by the PSST program. The best-fitted reconstruction of the observed dynamics was attained when the water in the cavity was assigned epsilon </= 55, corroborating the theoretical estimation of Sansom (Breed, J. R., I. D. Kerr, and M. S. P. Sansom. 1996. Biophys. J. 70:1643-1661). The dielectric constant calculated for reversed micelles of comparable size (Cohen, B., D. Huppert, K. M. Solntsev, Y. Tsfadia, E. Nachliel, and M. Gutman. 2002. JACS. 124:7539-7547) allows us to set a margin of epsilon = 50 +/- 5

A fast in silico simulation of ion flux through the large-pore channel proteins.

The PSST program (see accompanying article) utilizes the detailed structure of a large-pore channel protein as the sole input for selection of trajectories along which negative and positive ions propagate. In the present study we applied this program to reconstruct the ion flux through five large-pore channel proteins (PhoE, OmpF, the WT R. blastica general diffusion porin and two of its mutants). The conducting trajectories, one for positive and one for negative particles, are contorted pathways that run close to arrays of charged residues on the inner surface of the channel. In silico propagation of the charged particles yielded passage time values that are compatible with the measured average passage time of ions. The calculated ionic mobilities are close to those of the electrolyte solution of comparable concentrations. Inspection of the transition probabilities along the channel revealed no region that could impose a rate-limiting step. It is concluded that the ion flux is a function of the whole array of local barriers. Thus, the conductance of the large-pore channel protein is determined by the channel's shape and charge distribution, while the selectivity also reflects the features of the channel's vestibule

Proton transfer dynamics on the surface of the late M state of bacteriorhodopsin.

The cytoplasmic surface of the BR (initial) state of bacteriorhodopsin is characterized by a cluster of three carboxylates that function as a proton-collecting antenna. Systematic replacement of most of the surface carboxylates indicated that the cluster is made of D104, E161, and E234 (Checover, S., Y. Marantz, E. Nachliel, M. Gutman, M. Pfeiffer, J. Tittor, D. Oesterhelt, and N. Dencher. 2001. Biochemistry. 40:4281-4292), yet the BR state is a resting configuration; thus, its proton-collecting antenna can only indicate the presence of its role in the photo-intermediates where the protein is re-protonated by protons coming from the cytoplasmic matrix. In the present study we used the D96N and the triple (D96G/F171C/F219L) mutant for monitoring the proton-collecting properties of the protein in its late M state. The protein was maintained in a steady M state by continuous illumination and subjected to reversible pulse protonation caused by repeated excitation of pyranine present in the reaction mixture. The re-protonation dynamics of the pyranine anion was subjected to kinetic analysis, and the rate constants of the reaction of free protons with the surface groups and the proton exchange reactions between them were calculated. The reconstruction of the experimental signal indicated that the late M state of bacteriorhodopsin exhibits an efficient mechanism of proton delivery to the unoccupied-most basic-residue on its cytoplasmic surface (D38), which exceeds that of the BR configuration of the protein. The kinetic analysis was carried out in conjunction with the published structure of the M state (Sass, H., G. Büldt, R. Gessenich, D. Hehn, D. Neff, R. Schlesinger, J. Berendzen, and P. Ormos. 2000. Nature. 406:649-653), the model that resolves most of the cytoplasmic surface. The combination of the kinetic analysis and the structural information led to identification of two proton-conducting tracks on the protein's surface that are funneling protons to D38. One track is made of the carboxylate moieties of residues D36 and E237, while the other is made of D102 and E232. In the late M state the carboxylates of both tracks are closer to D38 than in the BR (initial) state, accounting for a more efficient proton equilibration between the bulk and the protein's proton entrance channel. The triple mutant resembles in the kinetic properties of its proton conducting surface more the BR-M state than the initial state confirming structural similarities with the BR-M state and differences to the BR initial state