Surfaces with Controllable Topography and Chemistry Used as a Template for Protein Crystallization

Abstract

Surfaces with controllable topography and chemistry were prepared to act as substrates for protein crystallization, in order to investigate the influence of these surface properties on the protein crystallization outcome. Three different methods were investigated to deposit 1,3,5-tris(10-carboxydecyloxy)benzene (TCDB) on a muscovite mica substrate to find the best route for controlled topography. Of these three, sublimation worked best. Contact angle measurements revealed that the surfaces with short exposure to the TCDB vapor (20 min or less) are hydrophilic, while surfaces exposed for 30 min or longer are hydrophobic. The hydrophilic surfaces are flat with low steps, while the hydrophobic surfaces contain macrosteps. Four model proteins were used for crystallization on the surfaces with controlled topography and chemistry. Hen egg white lysozyme crystals were less numerous on the surface with macrosteps than on smoother surfaces. On the other hand, insulin nucleated faster on the hydrophobic surfaces with macrosteps, and therefore, the crystals were more abundant and smaller. Bovine serum albumin and talin protein crystals were more numerous on all TCDB functionalized surfaces, compared to the reference clean muscovite mica surfaces. Overall, this shows that surface topography and chemistry is an important factor that partly determines the outcome in a protein crystallization experiment.

Figure 1

Ball and stick model of muscovite mica (A), and chemical structure of 1,3,5-tris(10-carboxydecyloxy)benzene (TCDB) (B).

Figure 2

AFM height image of drop casted TCDB structures on muscovite mica (A); cross section of the surface (B).

Figure 3

AFM height image of dip-coated TCDB on muscovite mica (A); cross section of the surface (B).

Figure 4

AFM height images of evaporated TCDB on muscovite mica after 6 min showing a flat substrate (A), 20 min showing a surface with low steps (B), and 60 min of growth showing a surface with macrosteps (C).

Figure 5

Number of insulin crystals on (functionalized) muscovite mica as a function of time.

Figure 6

Optical microscopy images of insulin crystals on TCDB-functionalized surfaces with 20 min. (A) and 60 min of evaporation (B). The scale bar indicates 100 μm in both images.

Figure 7

Number of HEWL crystals on (functionalized) muscovite mica as a function of time.

Figure 8

Number of talin crystals on (functionalized) muscovite mica as a function of time.

Figure 9

Number of bovine serum albumin crystals on (functionalized) muscovite mica. The nucleation rate is relatively fast, as no significant differences in the number of crystals are observed over time in the experiments with BSA.