Expanding the molecular recognition repertoire of antifreeze polypeptides: effects on nucleoside crystal growthw

Crystal growth control is essential in various fields of science and technology (e.g., chemistry, materials science, pharmaceutical development). The concept of molecular recognition has been successfully used to elucidate the effects of additives (foreign ions or molecules) on crystal growth. Peptides and proteins are often used in vivo and in vitro to control the growth of minerals and produce new forms of solids with different physicochemical properties. Size and shape control of organic crystals, however, is more difficult due to their anisotropic properties (different atomic arrangements in three dimensions). Antifreeze polypeptides (AFPs) are a structurally diverse group of proteins found in many cold-adapted organisms (e.g., fish, insect) to protect them from freeze damage in a noncolligative manner, providing an intriguing example of ice crystal growth control. AFPs bind to specific faces of ice crystals and modify the habit of the ice crystals. Their affinity to ice depends on hydrogen bonding and hydrophobic interactions, unlike most protein–mineral interactions where ionic interactions often play a dominant role. Ice and clathrate hydrates (ice-like crystalline solids) are known to be inhibited and modified by AFPs. Nevertheless, we speculate that the recognition ability of AFPs is beyond ice and ice-like crystalline solids based on the facts: (1) ice surfaces are not well ordered at the molecular level compared to other inorganic or organic crystal surfaces and (2) AFPs coexist with many other solutes in vivo (e.g., various low molecular solutes present with AFPs in the hemolymph of Dendroides canadensis) diminishing the chance of the growth of single ice crystals. In this study, we for the first time demonstrate that AFPs can efficiently inhibit the nucleation and modify the single crystal growth of 5-methyluridine (mU), cytidine (C), and inosine (I). mU, C, and I are widely used nucleosides in the pharmaceutical industry, but little is known about their size and shape control using additives. This study also presents a distinct example of effective control of nucleoside crystal growth by additives. We obtained the crystals of mU, C, and I, respectively, by evaporation of their aqueous solutions at room temperature (ESIw). The resulting crystals of the three nucleosides appear as orthorhombic needles (Fig. 1a; Fig. S2, and S4, ESIw). The effect of a beetle AFP from D. canadensis (DAFP-1) on mU crystal growth was first investigated. DAFP-1 is a b-helical repeat protein with a size of 9 kDa containing 8 disulfide bonds, which are important for its structure and function as antifreeze. DAFP-1 was studied previously in our laboratory for its antifreeze enhancement effect by small molecular enhancers and interactions between DAFP-1 and reduced nicotinamide adenine dinucleotide in solution have been demonstrated recently. To completely inhibit single crystal growth, a reasonable additive/nucleoside molar ratio (the critical ratio) is needed. Different amounts of DAFP-1 were added directly to mU solution to determine the critical ratio of DAFP-1/mU (ESIw). The direct addition of DAFP-1 at all the tested concentrations delayed the first appearance of mU precipitates in the solutions (Table S1, ESIw), while higher additive/mU molar ratios resulted in a more significant delay. The critical ratio of DAFP-1/mU was estimated to be 3.0 10 , where the ratio is 3.0 10 5 or higher, no single mU crystal was detected, but reed-like amorphous mU precipitates (Fig. 1b).