Structure Formation in Class†I and Class†II Hydrophobins at the Air-Water Interface under Multiple Compression/Expansion Cycles.

Hydrophobins are small amphiphilic fungal proteins empirically divided into two classes. We investigated the self-assembled structures of class I SC3 from S. commune and class II HFBII from T. reesei transferred to mica from the air-water interface by using the Langmuir-Schaefer (LS) technique and atomic force microscopy (AFM). The main focus is the influence of areal constraint and multiple compressions and expansions on the morphology of the protein films. SC3 shows a rather homogenous coverage of the mica surface, with fibrillary structures. Multiple compressions to a surface pressure of 13 mn m-1 led to a shortening of the fibrils. HFBII exhibits multilayered structures of varying thickness at higher surface pressures. Multiple compressions led to a variety of large, multilayer aggregates. Several compressions and expansions homogenized the films of both types. Both proteins showed similar dendritic structures with relevant length scales of at least several hundred nanometers at pressures of 13 mn m-1 and above, although the primary structures they assemble into are usually different in size and type, and range from fibrils to hexagonally ordered films. These dendritic structures may stem from a combination of mechanical influences, such as compressions, expansions, and the drying effect during LS transfer, which may simulate processes during physiological applications of hydrophobins, such as encapsulation or release of spores.

The cmc-value of a bolalipid with two phosphocholine headgroups and a C24 alkyl chain: Unusual binding properties of fluorescence probes to bolalipid aggregates.

Bolalipids with a long alkyl chain and two phosphocholine polar groups self-assemble in water into two different types of aggregate structures, namely helical nanofibers at low temperature and two types of micellar aggregates at higher temperature. We tried to determine the critical aggregation concentration (cac) or critical micellar concentration (cmc) of the bolalipid tetracosane-1,24-bis(phosphocholine) (PC-C24-PC) by using different fluorescent probes. The use of pyrene or pyrene derivatives as fluorophores failed, whereas the probes 1,8-ANS and particularly bis-ANS gave consistent results. The structure of the bolalipid aggregates obviously hinders partitioning or binding of pyrene derivatives into the micellar interior, whereas 1,8-ANS and bis-ANS can bind to the surface of the aggregate structures. The observed large increase in fluorescence intensity of bis-ANS indicates that binding to the hydrophobic surface of the aggregates leads to a reduction of the dye mobility. However, binding of bis-ANS is relatively weak, so that the determination of a cac/cmc-value is difficult. Simulations of the intensity curves for PC-C24-PC lead to estimates of the cac/cmc-value of 0.3-1.0×10-6M, depending on the structure of the aggregates. Single molecule fluorescence correlation spectroscopy was used to determine the mobility of bis-ANS as a function of concentration of PC-C24-PC. The dye diffusion time and the molecular brightness are lower at low bolalipid concentration, when only free dye is present, and increase at higher concentration when bis-ANS is bound to the aggregates. The experimental cac/cmc-values are higher than those estimated, using an incremental method for the change in Gibbs free energy for micellization with n-alkyl-phosphocholines with only one polar group as a comparison. Apparently, for PC-C24-PC in micellar or fibrous aggregates, more CH2 groups are exposed to water than in a conventional micelle of an n-alkyl-phosphocholine.