Laser ablation combined with Nanoimprint Lithography technology as new surface engineering approach to produce novel polymer-based heteronucleants for recalcitrant protein crystallization

Macromolecule crystallography is currently the most powerful technique for determining the three dimensional structure of proteins at atomic resolution. However, despite its enormous level of technical development achieved, it is still limited by the great difficulty of obtaining crystals that, after exposure to X-rays, allow a highresolution diffraction pattern from which the structure of the target protein can be elucidated. Even today, the crystallization process is a trial-and-error procedure that requires enormous experimental effort and high economic costs. In this research work, Laser Ablation technology in combination with NanoImprint Lithography was applied on Polycarbonate surfaces to produce novel heteronucleating agents containing multiscale surface topographies (in the micro- and submicro-scale) that promote the growth of protein crystals. Two human proteins, known to be recalcitrant to crystallization when using standard protocols, were used in the assays: (i) a construct of the CNNM4 magnesium transporter lacking amino acid residues 545-730 (HsCNNM4545-730) and (ii) cystathionine beta-synthase enzyme lacking residues 516-525 (HsCBS Delta 516-525). The surface engineering solution was validated by comparing the number of novel crystallization conditions induced by those surfaces compared against the control and the use of the Naomi's Nucleant, a commercially available heteronucleant made of mesoporous bioglass. The aspect ratio of the topography emerges as the topographical parameter that most contributes to protein growth on patterned surfaces. Additionally, the presence of a random distribution of micropores as well as a hierarchical (merging micro- and nano - scale features) surface topography also resulted in a more efficient crystallization process, showing an increment of between 38 and 68% in the number of new crystallization conditions, depending on the surface topography and protein selected. A conclusion is made that Laser Ablation with ultrashort pulses and the combination with Nanoimprint Lithography can be efficiently applied on polymeric substrates to produce a novel concept of heteronucleants that would bridge the gap to the development of universal nucleation agents.

Automated summary

Macromolecule crystallography is the most powerful technique for determining protein structure, but is limited by the difficulty of obtaining suitable crystals. This research explores the use of Laser Ablation and Nanoimprint Lithography to create novel heteronucleants on polymeric substrates, which promote protein crystal growth. The results show that the aspect ratio of the topography and the combination of micro- and nano-scale features significantly improve the efficiency of the crystallization process.