Biophysical characterization of the domain association between cytosolic A and B domains of the mannitol transporter enzymes IIMtl in the presence and absence of a connecting linker

The mannitol transporter enzyme II^Mtl of the bacterial phosphotransferase system is a multi‐domain protein that catalyzes mannitol uptake and phosphorylation. Here we investigated the domain association between cytosolic A and B domains of enzyme II^Mtl, which are natively connected in Escherichia coli, but separated in Thermoanaerobacter tengcongensis. NMR backbone assignment and residual dipolar couplings indicated that backbone folds were well conserved between the homologous domains. The equilibrium binding of separately expressed domains, however, exhibited ～28‐fold higher affinity compared to the natively linked ones. Phosphorylation of the active site loop significantly contributed to the binding by reducing conformational dynamics at the binding interface, and a few key mutations at the interface were critical to further stabilize the complex by hydrogen bonding and hydrophobic interactions. The affinity increase implicated that domain associations in cell could be maintained at an optimal level regardless of the linker.     

AB‐Bind: Antibody binding mutational database for computational affinity predictions

Antibodies (Abs) are a crucial component of the immune system and are often used as diagnostic and therapeutic agents. The need for high‐affinity and high‐specificity antibodies in research and medicine is driving the development of computational tools for accelerating antibody design and discovery. We report a diverse set of antibody binding data with accompanying structures that can be used to evaluate methods for modeling antibody interactions. Our Antibody‐Bind (AB‐Bind) database includes 1101 mutants with experimentally determined changes in binding free energies (ΔΔG) across 32 complexes. Using the AB‐Bind data set, we evaluated the performance of protein scoring potentials in their ability to predict changes in binding free energies upon mutagenesis. Numerical correlations between computed and observed ΔΔG values were low (r = 0.16–0.45), but the potentials exhibited predictive power for classifying variants as improved vs weakened binders. Performance was evaluated using the area under the curve (AUC) for receiver operator characteristic (ROC) curves; the highest AUC values for 527 mutants with |ΔΔG| > 1.0 kcal/mol were 0.81, 0.87, and 0.88 using STATIUM, FoldX, and Discovery Studio scoring potentials, respectively. Some methods could also enrich for variants with improved binding affinity; FoldX and Discovery Studio were able to correctly rank 42% and 30%, respectively, of the 80 most improved binders (those with ΔΔG < −1.0 kcal/mol) in the top 5% of the database. This modest predictive performance has value but demonstrates the continuing need to develop and improve protein energy functions for affinity prediction.     

Micronutrient Nutrition of Plants

Currently, there are eight trace elements considered to be essential for higher plants, Fe, Zn, Mn, Cu, Ni, B, Mo, and Cl. Possibly, other elements will be discovered to be essential because of recent advances in nutrient solution culture techniques and in the commercial availability of highly sensitive analytical instrumentation for elemental analysis. Much remains to be learned about the physiology of micronutrient absorption, translocation and deposition in plants, and about the functions they perform in plant growth and development. This review briefly summarizes the current knowledge of micronutrients in plants and than presents some new speculations on the mechanisms of micronutrient uptake and translocation in plants.     

Mechanistic simulation models of nutrient uptake: A review

Mechanistic models of nutrient uptake consider diffusion and mass flow acting simultaneously to supply nutrients to the sorbing root surface. Plant parameters that determine nutrient uptake include those describing changes in root geometry and size due to root growth and others describing kinetics of the nutrient uptake process. Mechanistic models generally assume that nutrient uptake occurs evenly along the roots that are uniformly distributed in homogeneous and isotropic soil having no temporal and spatial gradients in volumetric moisture content. Uptake of immobile nutrients (like P and K) is mainly determined by the soil-supply parameters and is well predicted by the simulation models. In contrast, uptake of mobile nutrients (e.g. Ca and Mg) that usually accumulate at the root surface is determined mainly by the plant-uptake parameters; prediction of uptake of those nutrients is subject to a much wider error due to uncertainties of applying kinetic parameters measured on hydroponically-grown plants to soil-grown plants. Comparison of model-predicted and experimentally-observed uptake values should be done by calculating the mean squares of deviates instead of performing regression analysis, especially if data that encompass a relatively wide range in root length are considered. Complementary-ion effects occurring at the soil-root interface raise the need for developing a multi-nutrient uptake model that will simultaneously calculate uptake of several essential nutrients taking into account interactions among them.     

An improved leaf-disk antifeedant bioassay and its application for the screening of Hokkaido plants

In the course of our search for new insect antifeedant substances from plants, we have devised an improved antifeedant leaf-disk bioassay. Our system allows an accurate measurement of consumed disk surfaces, using a video camera interfaced with a computer. The scanned image of the leaf disks is stored, and the eaten areas are measured with the help of a video-image analysis software. This new method allows for precise quantification of insect antifeedant activity tested on leaf material. This method was applied to the screening of Hokkaido plants for antifeedant activity. Out of 206 samples, seven demonstrated significant activity against larvae of the tobacco cutworm,Spodoptera litura.     

Anesthetic-protein interaction Random versus helix polylysine monolayers and interaction with 1-alkanols

Penetration of 1-alkanols into monolayers of hydrophobic polypeptides, poly(ε-benzyloxycarbonyl-l-lysine) and poly(ε-benzyloxycarbonyl-dl-lysine), was compared with their adsorption on the air/water interface in the absence of monolayers. The polypeptide prepared from l-lysine is generally considered to be in the α-helical form whereas dl-copolymer polypeptide contains random-coiled portions due to the structural incompatibility between the two isomers. The free energy of adsorption of 1-alkanols on the air/water interface at dilute concentrations was ?0.68 kcal·mol^?1 per methylene group and 0.15 kcal·mol^?1 for the hydroxyl group at 25°C. In the close-packed state, the surface area occupied by each molecule of 1-alkanols of varying carbon chain-lengths showed nearly a constant value of about 27.2 Ų, indicating perpendicular orientation of the alkanol molecules at the interface. About 75% of the water surface was covered by 1-butanol in this close-packed state. The mode of adsorption of 1-alkanols on the vacant air/water interface followed the Gibbs surface excess while the mode on the polypeptide membranes followed the Langmuir adsorption isotherm, indicating that the latter is characterized by the presence of a finite number of binding sites. The free energies of adsorption of 1-alkanols on the l-polymer monolayers were more negative than those on the vacant air/water interface and less negative than those on the dl-copolymer monolayers. Thus, the affinity of 1-alkanols to the interface was in the order of vacant air/water interface <l-polymer <dl-copolymer. The difference between the air/water interface and l-polymer was about 0.54 kcal·mol^?1 and that between l-polymer and dl-copolymer was 0.17 kcal·mol^?1 at 25°C: the adsorption of 1-alkanols to the dl-copolymer was favored compared to the l-polymer. The polar moieties of the backbone of the dl-copolymer may be exposed to the aqueous phase at the disordered portion. Dipole interaction between this portion and 1-alkanol molecules may account for the enhanced adsorption of the alkanols to the dl-copolymer.     

Interactions of basement membrane components

The binding of laminin, type IV collagen, and heparan sulfate proteoglycan to each other was assessed. Laminin binds preferentially to native type IV (basement membrane) collagen over other collagens. A fragment of laminin (M_r 600 000) containing the three short chains (M_r 200 000) but lacking the long chain M_r 400 000) showed the same affinity for type IV collagen as the intact protein. The heparan sulfate proteoglycan binds well to laminin and to type IV collagen. These studies show that laminin, type IV collagen and heparan sulfate proteoglycan interact with each other. Such interactions in situ may determine the structure of basement membranes.     

A method for determining concentration profiles at the soil-root interface by thin slicing rhizospheric soil

Summary A method is described for the determination of concentration gradients in the vicinity of plant roots. Plants are grown in small containers in which the roots are separated from the soil by a screen of nylon cloth. Root hairs but not roots penetrate the screen into the soil. In order to investigate the rhizospheric soil, the soil is frozen by liquid nitrogen and sliced into layers about 0.06 mm thick by means of a refrigerated microtome.