Dependence of phase transition uniformity on crystal sizes characterized using birefringence

Solid-solid phase transitions (SSPTs) have been widely observed in crystals of organic or inorganic small-molecules. Although SSPTs in macromolecular crystals have been reported, the majority involve local atomic changes, such as those induced by changes in hydration. SSPTs driven by large conformational changes, however, can be more difficult to characterize since they often significantly disrupt lattice packing interactions. Such drastic changes make the cooperativity of molecular motion at the atomic level less easily achieved and more dependent on intrinsic properties of the crystal that define lattice order. Here, we investigate the effect of crystal size on the uniformity of SSPT in thin plate-like crystals of the adenine riboswitch aptamer RNA (riboA) by monitoring changes in crystal birefringence upon the diffusion of adenine ligand. The birefringence intensity is directly related to molecular order and the concurrent changes to polarizability of molecules that results from structural changes throughout the phase transition. The riboA crystals were loosely grouped into three categories (small, medium, and large) based on the surface area of the crystal plates. The time width of transition increased as a function of crystal size, ranging from ∼13 s for small crystals to ∼40 s for the largest crystal. Whereas the transitions in small crystals (<10 μm²) were mostly uniform throughout, the medium and large crystals exhibited large variations in the time and width of the transition peak depending on the region of the crystal being analyzed. Our study provides insight into the spatiotemporal behavior of phase transitions in crystals of biological molecules and is of general interest to time-resolved crystallographic studies, where the kinetics of conformational changes may be governed by the kinetics of an associated SSPT.