The pressure-induced changes in 15N enriched HPr from Staphylococcus carnosus were investigated by two-dimensional (2D) heteronuclear NMR spectroscopy at pressures ranging from atmospheric pressure up to 200 MPa. The NMR experiments allowed the simultaneous observation of the backbone and side-chain amide protons and nitrogens. Most of the resonances shift downfield with increasing pressure indicating generalized pressure-induced conformational changes. The average pressure-induced shifts for amide protons and nitrogens are 0.285 ppm GPa−1 at 278 K and 2.20 ppm GPa−1, respectively. At 298 K the corresponding values are 0.275 and 2.41 ppm GPa−1. Proton and nitrogen pressure coefficients show a significant but rather small correlation (0.31) if determined for all amide resonances. When restricting the analysis to amide groups in the β-pleated sheet, the correlation between these coefficients is with 0.59 significantly higher. As already described for other proteins, the amide proton pressure coefficients are strongly correlated to the corresponding hydrogen bond distances, and thus are indicators for the pressure-induced changes of the hydrogen bond lengths. The nitrogen shift changes appear to sense other physical phenomena such as changes of the local backbone conformation as well. Interpretation of the pressure-induced shifts in terms of structural changes in the HPr protein suggests the following picture: the four-stranded β-pleated sheet of HPr protein is the least compressible part of the structure showing only small pressure effects. The two long helices a and c show intermediary effects that could be explained by a higher compressibility and a concomitant bending of the helices. The largest pressure coefficients are found in the active center region around His15 and in the regulatory helix b which includes the phosphorylation site Ser46 for the HPr kinase. This suggests that this part of the structure occurs in a number of different structural states whose equilibrium populations are shifted by pressure. In contrast to the surrounding residues of the active center loop that show large pressure effects, Ile14 has a very small proton and nitrogen pressure coefficient. It could represent some kind of anchoring point of the active center loop that holds it in the right place in space, whereas other parts of the loop adapt themselves to changing external conditions.

The apoplasmic permeability of ectomycorrhizal roots of intact Eucalyptus pilularis seedlings infected with Pisolithus tinctorius on aseptic agar plates was examined using the nonbinding fluorochrome 8-hydroxypyrene-1,3,6-trisulphonate and lanthanum ions in conjunction with anhydrous freeze substitution and dry sectioning. Most mycorrhizas formed in the air above the agar surface, and in these the sheath rapidly became nonwettable and impermeable to the fluorochrome but was nevertheless permeable to lanthanum ions. In a few mycorrhizas which developed in contact with the agar the sheath remained permeable to both tracers when fully developed. This increased hydrophobicity of the sheath in mycorrhizas in the air above the agar surface might be explained by deposition of hydrophobins, but nevertheless it still allows an apoplasmic pathway for radial movement of ions. Regardless of their sheath permeation both apoplasmic tracers were always found throughout the Hartig net and were arrested at the Casparian bands and suberin lamellae of the exodermis. It is concluded that the fluorochrome must have moved longitudinally along the Hartig net which is a region of higher permeability than the sheath. Casparian bands in the exodermis of ectomycorrhizal roots have similar properties to those in nonmycorrhizal roots in excluding solutes and their exclusion of lanthanum ions indicates that they are not permeable to ions. The data do not support the concept of a totally sealed apoplasmic exchange compartment, but the differential permeability suggests that the sheath might allow radial transfer of ions but block loss of sugars and organic molecules of similar size.