Precipitant-Free Crystallization of Protein Molecules Induced by Incision on Substrate

Abstract

Nucleation of protein crystals has been shown to be facilitated by substrates decorated with both nano-to micro-scale hierarchical undulations and spatially varying surface potential. In fact, on such surfaces, several proteins were found to crystallize without having to use any precipitant in contrast to all other homogeneous and heterogeneous systems in which precipitant is an essential ingredient for nucleation. While these surfaces were so patterned whole through the area that was brought in contact with the protein solution, it was not clear exactly to what extent the surfaces were required to be patterned to trigger nucleation without use of any precipitant. Here we show that a simple incision may be enough on an otherwise smooth surface for this purpose. In particular, the substrate used here is a smooth silicone film with its surface plasma oxidized to create a thin crust of silica. An incision is then generated on this surface using a sharp razor blade. The silica crust being brittle leads to random nano-microscopic undulations at the vicinity of the incision. These undulations along with surface charge can induce protein crystal nucleation without precipitant.

Precipitant-Free Crystallization of Lysozyme and Glucose Isomerase by Drying

“…Although Ghatak et al showed that lysozyme and proteinase K seemed to be crystallized by the evaporation of water at 20 • C, they argued that the crystals were obtained due to the effects of an incision on plasma oxidized polydimethylsiloxane substrate; they claimed that crystals were not obtained by concentration of proteins by evaporation of water. Moreover, they did not show the diffraction data of the crystals [13].…”

Protein Crystallization by Membrane-Assisted Technology

“…Ghatak et al , recently tested protein crystallization on patterned and electrically charged films. PDMS films with charges, surface wrinkles, and a combination of the two were tested in lysozyme crystallization.…”

“…This is advantageous because it enables crystals to form in the metastable region, where moderate supersaturation slows their growth and increases the chance of obtaining well-diffractive examples. 55 Ghatak et al 56,57 recently tested protein crystallization on patterned and electrically charged films. PDMS films with charges, surface wrinkles, and a combination of the two were tested in lysozyme crystallization.…”

Enhanced Protein Crystallization on Nafion Membranes Modified by Low-Cost Surface Patterning Techniques

“…ordered mesoporous silica with 4–20 nm average pore size, for a given relationship between the protein radius of gyration and specific pore diameter of the nucleant particle. They also hypothesized a further stabilization of the nuclei formed in the nucleant pores induced by the presence of specific chemical moieties (such as −OH, −NH 2 , or −CH 3 ) on the pore wall. , Ghatak et al , obtained protein crystals without the help of a precipitant by combining a wrinkled PDMS surface with an oxidation treatment. Recent efforts have been oriented toward the evaluation of topography and roughness effects on nucleation over nucleant surfaces without altering the surface chemistry.…”