Quartz crystal microbalance based on torsional piezoelectric resonators.

A quartz crystal microbalance (QCM) is described, which is based on a torsional resonator, rather than a conventional thickness-shear resonator. Typical applications are measurements of film thickness in the coating industry and monitoring of biofouling. The torsional QCM is about a factor of 100 less sensitive than the conventional QCM. On the other hand, it can probe film thicknesses in the range of hundreds of microns, which is impossible with the conventional QCM due to viscoelastic artifacts. Data acquisition and data analysis proceed in analogy to the conventional QCM. An indicator of the material's softness can be extracted from the bandwidth of the resonance. Within the small-load approximation, the frequency shift is independent of whether the sample is applied to the face or to the side of the cylinder. Details of the geometry matter if the viscoelastic properties of the sample are of interest.

Surface topography of the p3 and p6 annexin V crystal forms determined by atomic force microscopy.

Annexin V is a member of a family of structurally homologous proteins sharing the ability to bind to negatively charged phospholipid membranes in a Ca(2+)-dependent manner. The structure of the soluble form of annexin V has been solved by X-ray crystallography, while electron crystallography of two-dimensional (2D) crystals has been used to reveal the structure of its membrane-bound form. Two 2D crystal forms of annexin V have been reported to date, with either p6 or p3 symmetry. Atomic force microscopy has previously been used to investigate the growth and the topography of the p6 crystal form on supported phospholipid bilayers (Reviakine et al., 1998). The surface structure of the second crystal form, p3, is presented in this study, along with an improved topographic map of the p6 crystal form. The observed topography is correlated with the structure determined by X-ray crystallography.

Structural and functional role of the disulfide bridges in the hydrophobin SC3.

Hydrophobins function in fungal development by self-assembly at hydrophobic-hydrophilic interfaces such as the interface between the fungal cell wall and the air or a hydrophobic solid. These proteins contain eight conserved cysteine residues that form four disulfide bonds. To study the effect of the disulfide bridges on the self-assembly, the disulfides of the SC3 hydrophobin were reduced with 1,4-dithiothreitol. The free thiols were then blocked with either iodoacetic acid (IAA) or iodoacetamide (IAM), introducing eight or zero negative charges, respectively. Circular dichroism and infrared spectroscopy showed that after opening of the disulfide bridges SC3 is initially unfolded. IAA-SC3 did not self-assemble at the air-water interface upon shaking an aqueous solution. Remarkably, after drying down IAA-SC3 or after exposing it to Teflon, it refolded into a structure similar to that observed for native SC3 at these interfaces. Iodoacetamide-SC3 on the other hand, which does not contain extra charges, spontaneously refolded in water in the amyloid-like beta-sheet conformation, characteristic for SC3 assembled at the water-air interface. From this we conclude that the disulfide bridges of SC3 are not directly involved in self-assembly but keep hydrophobin monomers soluble in the fungal cell or its aqueous environment, preventing premature self-assembly.

Growth of Protein 2-D Crystals on Supported Planar Lipid Bilayers Imaged in Situ by AFM.

Theories of crystallization, both in 3-D and 2-D, are still very limited, mainly due to the scarcity of experimental approaches providing pertinent data on elementary phenomena. We present here a novel experimental approach for following, in real time and in situ, the process of 2-D crystallization of proteins on solid supports. Using annexin V as a model of a protein binding by affinity to a lipid matrix, we show that 2-D crystals of proteins can be formed on supported planar lipid bilayers (SPBs). Atomic Force Microscopy (AFM) enables the process of 2-D crystal growth to be visualized. The submolecular organization of the crystals was characterized at a resolution of approximately 2 nanometers, and defects, hitherto not observed in protein crystals, were resolved. These results have potential applications in basic and applied sciences. Copyright 1998 Academic Press.

Surfactosomes: a novel approach to the reconstitution and 2-D crystallisation of membrane proteins.

The formation of vesicle-like structures (termed surfactosomes) and lamellar sheets from solutions containing ammonium perfluoroocanoate (APFO) is illustrated using conventional and cryo-transmission electron microscopy. It is shown how this detergent can be used for the solubilisation, reconstitution and 2-D crystallisation of membrane proteins as demonstrated for the major protein of the membrane sector of the V-type H+-ATPase (16-kDa protein). Electron microscopical analysis of 2-D crystals of the 16-kDa protein (a=b=13.0+/-0.2nm with gamma = 90 degrees and p4 projection symmetry) revealed a unit cell comprising four dimeric complexes of the 16-kDa protein the significance of which is discussed.