We have measured the time-resolved adsorption kinetics of the mussel adhesive protein (Mefp-1) on a nonpolar, methyl-terminated (thiolated) gold surface, using three independent techniques: quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance, and ellipsometry. The QCM-D and ellipsometry data shows that, after adsorption to saturation of Mefp-1, cross-linking of the protein layer using NaIO4 transforms it from an extended (approximately 20 nm), water-rich, and hydrogel-like state to a much thinner (approximately 5 nm), compact, and less water-rich state. Furthermore, we show how quantitative data about the thickness, shear elastic modulus, and shear viscosity of the protein film can be obtained with the QCM-D technique, even beyond the Sauerbrey regime, if frequency (f) and energy dissipation (D) measurements measured at multiple harmonics are combined with theoretical simulations using a Voight-based viscoelastic model. The modeling result was confirmed by substituting H2O for D2O. As expected, the D2O substitution does not influence the actual adsorption behavior, but resulted in expected differences in the estimated effective density and shear viscosity. These results provide new insight and understanding about the adsorption kinetics and crosslinking behavior of Mefp-1. They also demonstrate how the above three techniques complement each other for biomolecule adsorption studies.

The phase behavior and microstructure of the two ternary systems monoolein (MO)−sodium oleate (NaO)−water (2H2O) and MO−oleic acid (OA)−2H2O are studied by a combination of optical microscopy, cryo-transmission electron microscopy, small-angle X-ray diffraction, and NMR methods. The results demonstrate significant differences in phase behavior between the two systems. The isothermal phase diagram of the MO−NaO−2H2O system is dominated by a large lamellar liquid crystalline phase that shows an ideal swelling up to high water contents. Stable vesicles are the dominant aggregates at water concentration >90 wt %. The existence of a lamellar phase is, however, absent from the MO−OA−2H2O system, where the largest single-phase region is a reversed hexagonal liquid crystalline phase, HII, at low water content. A similar water-poor HII phase is also identified for the MO−NaO−2H2O system. The two types of bicontinuous cubic structures, gyroid (CG) and diamond (CD), formed by the binary MO−2H2O system are also present in the ternary systems. Part of the single CG phase initially formed by the ternary system with NaO is found to be metastable and becomes destabilized within a few weeks, leaving the rest of the CG phase which is stable like other thermodynamically stable phases for the system. A cubic phase with a reversed micellar type structure is characterized for the oleic acid system. The experimentally determined phase diagrams and the phase structures can be qualitatively understood in terms of the geometry of the lipid molecule in combination with electrostatic effects.

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Cytochrome c (cyt c) has been studied as an example of a peripheral membrane protein that forms a cubic phase with monooleoylglycerol (monoolein, MO) and aqueous solutions. The unit cell dimensions of the cubic phases (space group Pn3m) in relation to the composition of the aqueous solution and protein concentration were analyzed according to the concept of a lipid packing parameter in the bilayer. The interaction between cyt c and MO in the cubic phase was studied by FT-IR spectroscopy and differential scanning calorimetry (DSC). The FT-IR data indicated a somewhat higher conformational order of the MO acyl chain and structural rearrangements of the polar head-group region in the cubic MO−cyt c−H2O phase. These findings, together with the increase in unit cell dimension, suggested a decrease of the MO packing parameter upon protein incorporation. Furthermore, a competitive interaction between the protein and buffer ions at the lipid bilayer was observed. DSC measurements showed that incorporation of cyt c into the cubic MO−H2O phase resulted in a significant decrease in temperature during the phase transitions cubic → reversed hexagonal → reversed micellar, and the unfolding of the protein took place simultaneously with the first phase transition. Additionally, voltammetric and chronoamperometric studies of the direct redox conversions of cyt c at the 4,4‘-dithiodipyridine-modified gold electrode revealed that the mobility of the protein molecules within the cubic phase was highly restricted. Altogether, these findings indicate that nonelectrostatic interactions between peripheral proteins and lipid molecules in membranes might also play a role in regulating biological function.

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