Structural evolution of protein-biofilms: Simulations and experiments

Abstract

The control of biofilm formation is a challenging goal that has not been reached yet in many aspects. One unsolved question is the role of van der Waals forces and another is the importance of mutual interactions between the adsorbing and the adsorbed biomolecules ("critical crowding"). In this study, a combined experimental and theoretical approach is presented, which fundamentally probes both aspects. On three model proteins-lysozyme, α-amylase, and bovine serum albumin-the adsorption kinetics is studied experimentally. Composite substrates are used enabling a separation of the short- and the long-range forces. Although usually neglected, experimental evidence is given for the influence of van der Waals forces on the protein adsorption as revealed by in situ ellipsometry. The three proteins were chosen for their different conformational stabilities in order to investigate the influence of conformational changes on the adsorption kinetics. Monte Carlo simulations are used to develop a model for these experimental results by assuming an internal degree of freedom to represent conformational changes. The simulations also provide data on the distribution of adsorption sites. By in situ atomic force microscopy we can also test this distribution experimentally, which opens the possibility to, e.g., investigate the interactions between adsorbed proteins.

Figure 1

ζ-potential of hydrophobic and hydrophilic silicon wafers with thin and thick oxide thickness as functions of pH. For the ζ-potential, differences in oxide layer thickness are irrelevant. Note that adsorption experiments in this study have been performed at pH 7.0 and 4.75 (vertical dashed lines) (in cooperation with Zimmermann, TU Dresden).

Figure 2

Schematics of a conformational change from a compact to an expanded state and vice versa using probabilities given by the Metropolis algorithm. In the expanded state, the particle has the shape of the ellipsoid with long axes of the length Y2a and a short axis with a length of Y^*2a=2a∕Y².

Figure 3

Simulation box.

Figure 4

Adsorption kinetics of (a) lysozyme and (b) amylase on the four different types of surfaces. Experiments are carried out in a 10 mM phosphate buffer of pH 7 at 37 °C.

Figure 5

BSA adsorption kinetics on hydrophobized silicon wafers with (a) thick and (b) thin silicon oxide layers. Measurements at room temperature and 37.5 °C are shown. A non-Langmuir-like adsorption kinetics can only be observed on wafers with thin oxide layers and the intermediate linear regime is elongated at room temperature.

Figure 6

Adsorption kinetics of simulations with (black) and without (red) change of conformation. For lower surface coverages, the expanded state is more stable than the compact one. Packing more particles to the surface, the contrary occurs.

Figure 7

The energy landscape of a particle approaching the surface for two different points in time. The isolines mark steps of 0.2; the white area has infinite potential energy (measured in k_BT).

Figure 8

The energy landscape of a particle approaching the surface: This is a plot of the potential energy for different lateral distances, as shown in Fig. 7b.

Figure 9

The influence of the particle deformation (denoted by Y) on the type of kinetics: Solid curves mark the total number of adsorbed particles and the dashed curves the fraction of particles in the expanded state.

Figure 10

The effect of linker molecules for a stabilization of a protein film: (a) Initial configuration in the MC simulations using the standard form of interactions. (b) Stationary configuration in the computer simulation after adding a short-ranged contribution to the particle-particle interactions to mimic the effect of a linker. (c) AFM image of an amylase film stabilized by glutaraldehyde. For a simple comparison with the simulations, a threshold has been applied to the height scale such that protein is black and substrate is white.

Figure 11

Series of consecutive AFM images (1 μm×1 μm) of BSA (20 μl of 0.1 mM protein solution in acetate buffer of pH 4.75 and ionic strength I=8 mM at room temperature) adsorbed onto mica taken at the same area (slight drift) under stopped flow conditions. The bright objects represent single proteins.