Molecular switch in signal transduction: Reaction paths of the conformational changes in ras p21

Conformational changes in ras p21 triggered by the hydrolysis of GTP play an essential role in the signal transduction pathway. The path for the conformational change is determined by molecular dynamics simulation with a holonomic constraint directing the system from the known GTP-bound structure (with the γ-phosphate removed) to the GDP-bound structure. The simulation is done with a shell of water molecules surrounding the protein. In the switch I region, the side chain of Tyr-32, which undergoes a large displacement, moves through the space between loop 2 and the rest of the protein, rather than on the outside of the protein. As a result, the charged residues Glu-31 and Asp-33, which interact with Raf in the homologous RafRBD–Raps complex, remain exposed during the transition. In the switch II region, the conformational changes of α2 and loop 4 are strongly coupled. A transient hydrogen bonding complex between Arg-68 and Tyr-71 in the switch II region and Glu-37 in switch I region stabilizes the intermediate conformation of α2 and facilitates the unwinding of a helical turn of α2 (residues 66–69), which in turn permits the larger scale motion of loop 4. Hydrogen bond exchange between the protein and solvent molecules is found to be important in the transition. Possible functional implications of the results are discussed.

A Calcium-Selective Channel from Root-Tip Endomembranes of Garden Cress1

A Ca2+ channel from root-tip endomembranes of garden cress (Lepidium sativum L.) (LCC1) was characterized using the planar lipid-bilayer technique. Investigation of single-channel recordings revealed that LCC1 is voltage gated and strongly rectifying. In symmetrical 50 mm CaCl2 solutions, the single-channel conductance was 24 picosiemens. LCC1 showed a moderate selectivity for Ca2+ over K+ (9.4:1) and was permeable for a range of divalent cations (Ca2+, Ba2+, and Sr2+). In contrast to Bryonia dioica Ca2+ channel 1, a Ca2+-selective channel from the endoplasmic reticulum of touch-sensitive tendrils, LCC1 showed no bursting channel activity and had a low open probability and mean open time (2.83 ms at 50 mV). Inhibitor studies demonstrated that LCC1 is blocked by micromolar concentrations of erythrosin B (inhibitor concentration for 50% inhibition [IC50] = 1.8 μm) and the trivalent cations La3+ (IC50 = 5 μm) and Gd3+ (IC50 = 10 μm), whereas verapamil showed no blocking effect. LCC1 may play an important role in the regulation of the cytoplasmic free Ca2+ concentration in root-tip and/or root-cap cells. The question of whether this ion channel is part of the gravitropic signal transduction pathway deserves further investigation.

Co-Permeability of 3H-Labeled Water and 14C-Labeled Organic Acids across Isolated Plant Cuticles1 : Investigating Cuticular Paths of Diffusion and Predicting Cuticular Transpiration

Penetration of 3H-labeled water (3H2O) and the 14C-labeled organic acids benzoic acid ([14C]BA), salicylic acid ([14C]SA), and 2,4-dichlorophenoxyacetic acid ([14C]2,4-D) were measured simultaneously in isolated cuticular membranes of Prunus laurocerasus L., Ginkgo biloba L., and Juglans regia L. For each of the three pairs of compounds (3H2O/[14C]BA, 3H2O/[14C]SA, and 3H2O/[14C]2,4-D) rates of cuticular water penetration were highly correlated with the rates of penetration of the organic acids. Therefore, water and organic acids penetrated the cuticles by the same routes. With the combination 3H2O/[14C]BA, co-permeability was measured with isolated cuticles of nine other plant species. Permeances of 3H2O of all 12 investigated species were highly correlated with the permeances of [14C]BA (r2 = 0.95). Thus, cuticular transpiration can be predicted from BA permeance. The application of this experimental method, together with the established prediction equation, offers the opportunity to answer several important questions about cuticular transport physiology in future investigations.