Barbara Poliks

Plant Cell-Wall Cross-Links by REDOR NMR Spectroscopy

We present a new method that integrates selective biosynthetic labeling and solid-state NMR detection to identify in situ important protein cross-links in plant cell walls. We have labeled soybean cells by growth in media containing l-[ring-d(4)]tyrosine and l-[ring-4-(13)C]tyrosine, compared whole-cell and cell-wall (13)C CPMAS spectra, and examined intact cell walls using (13)C{(2)H} rotational echo double-resonance (REDOR) solid-state NMR. The proximity of (13)C and (2)H labels shows that 25% of the tyrosines in soybean cell walls are part of isodityrosine cross-links between protein chains. We also used (15)N{(13)C} REDOR of intact cell walls labeled by l-[ε-(15)N,6-(13)C]lysine and depleted in natural-abundance (15)N to establish that the side chains of lysine are not significantly involved in covalent cross-links to proteins or sugars.

Rotational-echo double-resonance NMR-restrained model of the ternary complex of 5-enolpyruvylshikimate-3-phosphate synthase.

The 46-kD enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate-3-phosphate (S3P) and phosphoenolpyruvate to form EPSP. The reaction is inhibited by N-(phosphonomethyl)-glycine (Glp), which, in the presence of S3P, binds to EPSP synthase to form a stable ternary complex. We have used solid-state NMR and molecular modeling to characterize the EPSP synthase–S3P–Glp ternary complex. Modeling began with the crystal coordinates of the unliganded protein, published distance restraints, and information from the chemical modification and mutagenesis literature on EPSP synthase. New inter-ligand and ligand-protein distances were obtained. These measurements utilized the native 31P in S3P and Glp, biosynthetically 13C-labeled S3P, specifically 13C and 15N labeled Glp, and a variety of protein-15N labels. Several models were investigated and tested for accuracy using the results of both new and previously published rotational-echo double resonance (REDOR) NMR experiments. The REDOR model is compared with the recently published X-ray crystal structure of the ternary complex, PDB code 1G6S. There is general agreement between the REDOR model and the crystal structure with respect to the global folding of the two domains of EPSP synthase and the relative positioning of S3P and Glp in the binding pocket. However, some of the REDOR data are in disagreement with predictions based on the coordinates of 1G6S, particularly those of the five arginines lining the binding site. We attribute these discrepancies to substantive differences in sample preparation for REDOR and X-ray crystallography. We applied the REDOR restraints to the 1G6S coordinates and created a REDOR-refined xray structure that agrees with the NMR results.

Molecular Dynamics simulations of thermal conductivity in composites consisting of aluminum oxide nanoparticles surrounded by polyethylene oxide

Molecular Dynamics (MD) simulations of heat flow in the composite systems consisting of aluminum oxide nanostructures surrounded by polyethylene oxide were performed using known forcefields with consistent treatment of covalent (polymer) and ionic (nanoparticles) components. A reverse non-equilibrium molecular dynamics (RNEMD) method [implemented in open source MD Simulator LAMMPS was utilized to impose a temperature gradient and obtain the values of thermal conductivity. Several simulation boxes containing layers (4 nm and 20 nm width) and spheres (3 nm and 6 nm radii) of aluminum oxide surrounded by polyethylene oxide have been built, equilibrated and subjected to RNEMD. The sizes of the boxes varied from 10/15 nm × 10/15 nm × 45/200 nm. The boxes contained 0.6*106 to 3*106 atoms. An enhancement of effective thermal conductivity from 0.3 W/m·K (for pure polymer) up to 1.1 W/m·K was achieved for the composites containing multiple 20 nm layers of aluminum oxide. The value of interfacial thermal resistance at the aluminum oxide/polymer interface obtained from the simulations was approximately 5*10-9 m2K/W. Temperature profiles from RNEMD atomistic simulations were compared to known bulk models. Patterns of time averaged local heat flux in different components of the composite systems were calculated.