Self-assembled monolayers as a tool to investigate the effect of surface chemistry on protein nucleation

Modified surfaces like siliconized glass are commonly used to support protein crystallization and facilitate obtaining crystals. Over the years, various surfaces have been proposed to decrease the energetic penalty required for consistent protein clustering, but scarce attention has been paid to the underlying mechanisms of interactions. Here, we propose self-assembled monolayers that are surfaces exposing fine-tuned moieties with a very regular topography and subnanometer roughness, as a tool to unveil the interaction between proteins and functionalized surfaces. We studied the crystallization of three model proteins having progressively narrower metastable zones, i.e., lysozyme, catalase, and proteinase K, on monolayers exposing thiol, methacrylate, and glycidyloxy groups. Thanks to comparable surface wettability, the induction or the inhibition of nucleation was readily attributed to the surface chemistry. For example, thiol groups strongly induced the nucleation of lysozyme thanks to electrostatic pairing, whereas methacrylate and glycidyloxy groups had an effect comparable to unfunctionalized glass. Overall, the action of surfaces led to differences in nucleation kinetics, crystal habit, and even crystal form. This approach can support the fundamental understanding of the interaction between protein macromolecules and specific chemical groups, which is crucial for many technological applications in the pharmaceutical and food industry.