High-quality single crystals of protein are indispensable for determining the three-dimensional structure of protein by X-ray crystallography. However, the production of such high-quality protein crystals is still challenging because nucleation of proteins generally occurs at very high supersaturation. We previously reported a novel technique to induce nucleation at low supersaturation by focused femtosecond laser irradiation and obtained high-quality protein crystals of various proteins.¹⁾ We revealed that laser-induced cavitation bubbles transiently and locally concentrate a supersaturated solution.²⁾ We are now investigating the detailed correlation between cavitation bubble behavior and protein nucleation.³⁾ In this study, we studied the effect of gel-solution interfaces on the femtosecond laser-induced nucleation of proteins.

 Supersaturated solutions of hen egg white lysozyme (HEWL) were pipetted onto agarose gel and irradiated with femtosecond laser pulses (800 nm, 200 fs). We found that the nucleation efficiency could be modulated by the distance between the laser focus and agarose gel surfaces (h). In particular, laser irradiation near the gel surface (h = 50 mm) could induce nucleation at very low supersaturation, where no nucleation could be induced in the bulk solutions even with femtosecond laser irradiation (h = 200 mm) (Fig. 1). Such enhancement of the nucleation was also confirmed for the membrane protein, acriflavine resistance protein B (AcrB). To gain further insights into the role of gel-solution interfaces, we conducted fast imaging of cavitation bubbles. We found that the cavitation bubbles shrank asymmetrically and then finally collapsed toward the solution side (Fig. 2b, h = 50 mm). This is in contrast to the symmetric collapse in bulk solutions (Fig. 2a, h = 200 mm), which generates a large amount heat and chemical decomposition at the focus⁴⁾. In addition, cavitation bubble behavior could be modulated by the gel concentration. These results indicate that the use of gel-solution interfaces is a successful method for the control of cavitation bubbles and can be cues to achieve effective nucleation. In this presentation, we will also discuss the correlation between the gel concentration and nucleation efficiency.

                    Fig. 1. Nucleation probabilities for HEWL on agarose gel.

                    Fig. 2. Microscopic fast imaging of cavitation bubbles.

Reference
1) H. Adachi et al., Jpn. J. Appl. Phys. 42 (2003) L798.
2) H. Y. Yoshikawa et al., J. Cryst. Growth 311 (2009) 956.
3) S. Nakayama et al., Cryst. Growth Des 13 (2013) 1491.
4) K. S. Suslick et al., Annu. Rev. Mater. Sci. 29 (1999) 295.

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