On Protein Crystal Nucleation Precursors

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.

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Chemical Engineering

Grantor

University of Houston

Year dc:date.issued

2017

Author dc:creator

• Byington, Michael

Advisors dc:contributor.advisor

• Vekilov, Peter G.
• Conrad, Jacinta C.

Committee members dc:contributor.committeemember

• Bark, Steven J.
• Lubchenko, Vassiliy
• Nikolaou, Michael
• Rimer, Jeffrey D.

• Protein crystal nucleation
• Image morphology
• Process control
• Microfluidics

Statement dc:rights

• The author of this work is the copyright owner. UH Libraries and the Texas Digital Library have their permission to store and provide access to this work. UH Libraries has secured permission to reproduce any and all previously published materials contained in the work. Further transmission, reproduction, or presentation of this work is prohibited except with permission of the author(s).

Language dc:language.iso

eng

Handle dc:identifier.uri

https://hdl.handle.net/10657/4788

OAI identifier oai:identifier

oai:uh-ir.tdl.org:10657/4788