On Protein Crystal Nucleation Precursors



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Protein crystallization is central to our understanding of molecular structures in biology, vital to industrial processes in the pharmaceutical industry, and increasingly appreciated as a critical block in understanding numerous disease pathologies. Although the nucleation process is critical in determining the nucleation rate and polymorph of the crystal, it is a poorly understood phenomenon. Here, we present the effects of shear flow on protein pre-nucleation clusters, which grow in size but dramatically decrease in number density. The similarity of the effect of shear and denaturants on the clusters suggest that partial unfolding of lysozyme is a crucial step in the mechanism of cluster formation. However, opposing effects on the clusters were observed with shear and reducing agents indicating that the shear effects are not the result of disulfide bridge destruction. Up until now, the dimers of the domain swapped dimer mechanism have been suggested by theory and consistent with experimental results but they have not been directly detected. Here, we present the first direct evidence of transient lysozyme dimers in solution via electrospray mass spectrometry. By inspecting the Fourier transform of the isotopic envelope of lysozyme in ammonium acetate buffer, we can show near the noise limit there is a species with double the mass and double the charge.
Work on process control to improve the performance of a microfluidic heater has been presented as well. The modification of PID control for the induction of a step change in the temperature using a resistive heater is demonstrated to lower the response time by an order of magnitude. This approach has broad application when designing controllers with the primary function of inducing step changes where the error signal is initially dominated by the set point change in manner that is repeated many times. Finally, we present an algorithm for single particle tracking to overcome challenges in image quality (low contrast, high particle density) and video frame rate. Although this algorithm was originally designed for quantifying the motility of breast cancer cells, single particle tracking has application in colloidal crystallization where we hope to apply them in future work.



Protein crystal nucleation, Image morphology, Process control, Microfluidics


Portions of this document appear in: Byington, Michael C., Mohammad S. Safari, Jacinta C. Conrad, and Peter G. Vekilov. "Protein conformational flexibility enables the formation of dense liquid clusters: tests using solution shear." The journal of physical chemistry letters 7, no. 13 (2016): 2339-2345.