Effect of nucleants in photothermally assisted crystallization

Abstract

Laser-induced crystallization is emerging as a promising technique to crystallize biomolecules like amino acids and proteins. The use of external materials as nucleants and novel seeding methods open new paths for protein crystallization. We report here the results of experiments that explore the effect of nucleants on laser-based crystallization of microlitre droplets of small molecules, amino acids, and proteins. The role of parameters like solute concentration, droplet volume, type and size of the nucleant, and laser power, are systematically investigated. In addition to crystallization of standard molecules like NaCl, KCl, and glycine, we demonstrate the crystallization of negatively (l-histidine), and positively (l-aspartic acid) charged amino acids and lysozyme protein. Single crystal X-ray diffraction and Raman spectroscopy studies unequivocally indicate that the nucleants do not alter the molecular structure of glycine, hydrogen bonding patterns, and packing. Localized vaporization of the solvent near the nucleant due to photothermal heating has enabled us to achieve rapid crystallization - within 3 s - at laser intensities of 0.1 MW cm, significantly lower than those reported earlier, with both saturated and unsaturated solutions. The outcome of the current experiments may be of utility in tackling various crystallization problems during the formation of crystals large enough to perform X-ray crystallography.

Effect of biocompatible nucleants in rapid crystallization of natural amino acids using a CW Nd:YAG laser

“…It should be noted that in some of the amino acids - like Gln, Phe - we observed both formation and dissolution of crystals; both seem to be prevalent phenomena 15 , 25 .…”

“…In our previous report 15 , we had reported crystallization of glycine molecules ranging from 1M (75 mg/ml) – 3M (225 mg/ml) concentration using different nucleants. Aluminum wire, as well as aluminum particles, took more or less similar times (3 s) in those studies, which is comparable to the values measured in our current experiments.…”

“…In such studies, the crystallization targets are usually dissolved in either D 2 O or aged solutions are used to obtain crystals upon laser irradiation. In our previous report, we have summarized the results of laser-induced crystallization with the pertinent literature 15 . We have also shown that use of nucleants in laser-induced crystallization drastically reduces both the laser intensity that is required as well as the overall crystallization time.…”

“…This work showed that laser-induced crystallization is highly useful in obtaining crystals of small molecules, including chalcone compounds, which are otherwise difficult to crystallize by conventional crystallization methods 14 . We have also used simple but highly effective nucleants with a CW Nd:YAG (λ = 1064 nm) laser to crystallize NaCl, KCl, glycine, aspartic acid, and histidine amino acids, and lysozyme protein 15 . This work has provided evidence that laser-facilitated, nucleants-assisted crystallization may offer the important advantage of very low incident laser power and the consequent non-alteration of biomolecule structures.…”

“…We recently reported the use of aluminum nucleants in conjunction with a laser to crystallize glycine, aspartic acid, and histidine amino acids on time scales as short as a few seconds 15 . In comparison with other nucleants, aluminum yielded significant time reduction and produced consistently positive results.…”