Characterizing Protein Crystal Nucleation

Characterizing Protein Crystal Nucleation A dissertation presented to the Faculty of the Graduate School of Arts and Sciences of Brandeis University, Waltham, Massachusetts by Sathish V. Akella We developed an experimental microfluidic based technique to measure the nucleation rates and successfully applied the technique to measure nucleation rates of lysozyme crystals. The technique involves counting the number of samples which do not have crystals as a function of time. Under the assumption that nucleation is a Poisson process, the fraction of samples with no crystals decays exponentially with the decay constant proportional to nucleation rate and volume of the sample. Since nucleation is a random and rare event, one needs to perform measurements on large number of samples to obtain good statistics. Microfluidics offers the solution of producing large number of samples at minimal material consumption. Hence, we developed a microfluidic method and measured nucleation rates of lysozyme crystals in supersaturated protein drops, each with volume of ≈ 1 nL. Classical Nucleation Theory (CNT) describes the kinetics of nucleation and predicts the functional form of nucleation rate in terms of the thermodynamic quantities involved, such as supersaturation, temperature, etc. We analyzed the measured nucleation rates in the context of CNT and obtained the activation energy and the kinetic pre-factor characterizing the nucleation process. One conclusion is that heterogeneous nucleation dominates crystallization. We report preliminary studies on selective enhancement of nucleation in one of the crystal polymorprhs of lysozyme (spherulite) using amorphous mesoporous bioactive gel-glass[48, 49], CaO.P2O5.SiO2 (known as bio-glass) with 2−10 nm pore-size diameter distribution. The pores act as heterogeneous nucleation centers and claimed[46] vi to enhance the nucleation rates by molecular confinement. The measured kinetic profiles of crystal fraction of spherulites indicate that the crystallization of spherulites may be proceeding via secondary nucleation pathways.