Protein Crystallization Methods and Apparatus

Protein Crystallization Methods and Apparatus

Bibliographic Details
Main Author: Ogbuoji, Ebuka
Language:English
Published: University of Toledo / OhioLINK 2019
Subjects:
Chemical Engineering
Biochemistry
Biology
Protein crystallization
methods
apparatus
dynamic light scattering
microgravity crystallization
Online Access:http://rave.ohiolink.edu/etdc/view?acc_num=toledo1556924307276754
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spelling ndltd-OhioLink-oai-etd.ohiolink.edu-toledo15569243072767542021-10-02T05:10:33Z Protein Crystallization Methods and Apparatus Ogbuoji, Ebuka Chemical Engineering Biochemistry Biology Protein crystallization methods apparatus dynamic light scattering microgravity crystallization Adequate knowledge of the protein structure at the atomic level facilitates understanding of its molecular function. Structural information of proteins can be obtained by X-ray (XRD) and neutron diffraction (ND) studies. Neutron diffraction provides information on placement of protons (hydrogen and deuterium) whereas this important structural information is missing in most XRD protein structures. Due to the relatively low flux of neutron sources, ND requires large volume crystals (~1mm3) for diffraction studies. Obtaining protein crystals with such large volume is not a trivial task and is largely dependent on optimizing crystal nucleation and growth rates in protein solutions. Both kinetic processes are dependent on supersaturation. Some solution additives can allow nucleation and growth at lower supersaturation levels to yield ND quality crystals.Dynamic light scattering (DLS) was used to assess the effect of some additives including acetonitrile (ACN), ethylene glycol (EG) and glycerol (GLY) on protein solubility at various temperatures by obtaining a diffusion interaction parameter kD which is closely related to the second viral coefficient (B22). Three proteins xylose isomerase (XI), hen egg white lysozyme (HEWL) and tryptophan synthase (TS) were used in this study. Itwas observed that at higher temperatures (≥18oC), the some of these additives acted as solubility enhancers.Another factor that affects crystal growth is the convective transport of precipitant solution in crystallization experiments caused by gravitational forces. Convection can lead to crystal imperfections and impede the growth of ND quality crystals. Microgravity crystallization of tryptophan synthase was conducted on the International Space Station (ISS) in an attempt to improve crystal morphology, quality and size. Additives were used in both microgravity and ground control experiments. It was observed that crystals with larger volume and better morphology were grown in microgravity compared to their ground based counterpart.A counter diffusion capillary dialysis apparatus was developed and used for crystallization of TS in both microgravity and ground environments. Equilibration time of common precipitants including PEG 400, 4000, 8000, MPD and ammonium sulfate in the capillary were obtained experimentally in horizontal orientation (capillary parallel to the gravitational field) and vertical orientation (perpendicular to the gravitational field). It was observed that precipitants equilibrated much faster in the horizontal orientation than the vertical orientation. Equilibration was hypothesized to be a diffusion driven process for capillaries in the vertical orientation. An analytical diffusive model and experimental data for precipitant equilibration in the vertical orientation showed good agreement which supports the hypothesis. 2019 English text University of Toledo / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=toledo1556924307276754 http://rave.ohiolink.edu/etdc/view?acc_num=toledo1556924307276754 unrestricted This thesis or dissertation is protected by copyright: some rights reserved. It is licensed for use under a Creative Commons license. Specific terms and permissions are available from this document's record in the OhioLINK ETD Center.
collection NDLTD
language English
sources NDLTD
topic Chemical Engineering
Biochemistry
Biology
Protein crystallization
methods
apparatus
dynamic light scattering
microgravity crystallization
spellingShingle Chemical Engineering
Biochemistry
Biology
Protein crystallization
methods
apparatus
dynamic light scattering
microgravity crystallization
Ogbuoji, Ebuka
Protein Crystallization Methods and Apparatus
author Ogbuoji, Ebuka
author_facet Ogbuoji, Ebuka
author_sort Ogbuoji, Ebuka
title Protein Crystallization Methods and Apparatus
title_short Protein Crystallization Methods and Apparatus
title_full Protein Crystallization Methods and Apparatus
title_fullStr Protein Crystallization Methods and Apparatus
title_full_unstemmed Protein Crystallization Methods and Apparatus
title_sort protein crystallization methods and apparatus
publisher University of Toledo / OhioLINK
publishDate 2019
url http://rave.ohiolink.edu/etdc/view?acc_num=toledo1556924307276754
work_keys_str_mv AT ogbuojiebuka proteincrystallizationmethodsandapparatus
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