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The surface of polyhydroxybutyrate (PHB) storage granules in bacteria is covered mainly by proteins referred to as phasins. The layer of phasins stabilizes the granules and prevents coalescence of separated granules in the cytoplasm and nonspecific binding of other proteins to the hydrophobic surfaces of the granules. Phasin Pha[P1.sub.Reu] is the major surface protein of PHB granules in Ralstonia eutropha H16 and occurs along with three homologues (PhaP2, PhaP3, and PhaP4) that have the capacity to bind to PHB granules but are present at minor levels. All four phasins lack a highly conserved domain but share homologous hydrophobic regions. To identify the region of [Pha[P1.sub.Reu] which is responsible for the binding of the protein to the granules, N-terminal and C-terminal fusions of enhanced green fluorescent protein with [Pha[P1.sub.Reu] or various regions of [Pha[P1.sub.Reu] were generated by recombinant techniques. The fusions were localized in the cells of various recombinant strains by fluorescence microscopy, and their presence in different subcellular protein fractions was determined by immunodetection of blotted proteins. The fusions were also analyzed to determine their capacities to bind to isolated PHB granules in vitro. The results of these studies indicated that unlike the phasin of Rhodococcus ruber, there is no discrete binding motif; instead, several regions of [Pha[P1.sub.Reu] contribute to the binding of this protein to the surface of the granules. The conclusions are supported by the results of a small-angle X-ray scattering analysis of purified [Pha[P1.sub.Reu], which revealed that [Pha[P1.sub.Reu] is a planar, triangular protein that occurs as trimer. This study provides new insights into the structure of the PHB granule surface, and the results should also have an impact on potential biotechnoiogical applications of phasin fusion proteins and PHB granules in nanobiotechnology.

Folded protein stabilization or destabilization induced by cosolvent in mixed aqueous solutions has been studied by differential scanning microcalorimetry and related to difference in preferential solvation of native and denatured states. In particular, the thermal denaturation of a model system formed by lysozyme dissolved in water in the presence of the stabilizing cosolvent glycerol has been considered. Transition temperatures and enthalpies, heat capacity, and standard free energy changes have been determined when applying a two-state denaturation model to microcalorimetric data. Thermodynamic parameters show an unexpected, not linear, trend as a function of solvent composition; in particular, the lysozyme thermodynamic stability shows a maximum centered at water molar fraction of about 0.6. Using a thermodynamic hydration model based on the exchange equilibrium between glycerol and water molecules from the protein solvation layer to the bulk, the contribution of protein-solvent interactions to the unfolding free energy and the changes of this contribution with solvent composition have been derived. The preferential solvation data indicate that lysozyme unfolding involves an increase in the solvation surface, with a small reduction of the protein-preferential hydration. Moreover, the derived changes in the excess solvation numbers at denaturation show that only few solvent molecules are responsible for the variation of lysozyme stability in relation to the solvent composition. [ABSTRACT FROM AUTHOR]

In solution small-angle neutron scattering has been used to study the solvation properties of lysozyme dissolved in water/glycerol mixtures. To detect the characteristics of the protein-solvent interface, 35 different experimental conditions (i.e., protein concentration, water/glycerol fraction in the solvent, content of deuterated compounds) have been considered and a suitable software has been developed to fit simultaneously the whole set of scattering data. The average composition of the solvent in the close vicinity of the protein surface at each experimental condition has been derived. In all the investigated conditions, glycerol resulted especially excluded from the protein surface, confirming that lysozyme is preferentially hydrated. By considering a thermodynamic hydration model based on an equilibrium exchange between water and glycerol from the solvation layer to the bulk, the preferential binding coefficient and the excess solvation number have been estimated. Results were compared with data previously derived for ribonuclease A in the same mixed solvent: even if the investigated solvent compositions were very different, the agreement between data is noticeable, suggesting that a unique mechanism presides over the preferential hydration process. Moreover, the curve describing the excess solvation number as a function of the solvent composition shows the occurrence of a region of maximal hydration, which probably accounts for the changes in protein stability detected in the presence of cosolvents. [ABSTRACT FROM AUTHOR]

This study presents NMR signal detection by means of a superconducting channel consisting of a Nb surface detection coil inductively coupled to a YBCO mixed sensor. The NMR system operates at a low-field (8.9 mT) in a magnetically shielded room suitable for magnetoencephalographic (MEG) recordings. The main fleld is generated by a compact solenoid and the geometry of the pick-up coil has been optimized to provide high spatial sensitivity [ABSTRACT FROM AUTHOR]