Automated high throughput protein crystallization screening at nanoliter scale and protein structural study on lactate dehydrogenase

dc.contributor.advisor Edward S. Yeung
dc.contributor.author Li, Fenglei
dc.contributor.department Chemistry
dc.date 2018-08-24T22:01:19.000
dc.date.accessioned 2020-06-30T08:14:10Z
dc.date.available 2020-06-30T08:14:10Z
dc.date.copyright Sun Jan 01 00:00:00 UTC 2006
dc.date.issued 2006-01-01
dc.description.abstract <p>The purposes of our research were: (1) To develop an economical, easy to use, automated, high throughput system for large scale protein crystallization screening. (2) To develop a new protein crystallization method with high screening efficiency, low protein consumption and complete compatibility with high throughput screening system. (3) To determine the structure of lactate dehydrogenase complexed with NADH by x-ray protein crystallography to study its inherent structural properties;Firstly, we demonstrated large scale protein crystallization screening can be performed in a high throughput manner with low cost, easy operation. The overall system integrates liquid dispensing, crystallization and detection and serves as a whole solution to protein crystallization screening. The system can dispense protein and multiple different precipitants in nanoliter scale and in parallel. A new detection scheme, native fluorescence, has been developed to form a two-detector system with a visible light detector for detecting protein crystallization screening results. This detection scheme can eliminate common false positives by distinguishing protein crystals from inorganic crystals in a high throughput and non-destructive manner. The entire system from liquid dispensing, crystallization to crystal detection is essentially parallel, high throughput and compatible with automation. The system was successfully demonstrated by lysozyme crystallization screening;Secondly, we developed a new crystallization method with high screening efficiency, low protein consumption and compatibility with automation and high throughput. A gas permeable membrane is employed to achieve the gentle evaporation required by protein crystallization. Protein consumption is significantly reduced to nanoliter scale for each condition and thus permits exploring more conditions in a phase diagram for given amount of protein. The protein crystals gotten by this method were experimentally proven to possess high x-ray diffraction qualities;Finally, we crystallized human lactate dehydrogenase 1 (H4) complexed with NADH and determined its structure by x-ray crystallography;The structure of LDH/NADH displays open, closed conformation or two-conformation on the active site while the subunits in LDH/NADH/inhibitor are all in close conformation. Difference in subunit conformation was observed among the structures independently solved from multiple individual LDH/NADH crystals;Structural differences observed among crystals suggest the existence of multiple conformers in solution.</p>
dc.format.mimetype application/pdf
dc.identifier archive/lib.dr.iastate.edu/rtd/1924/
dc.identifier.articleid 2923
dc.identifier.contextkey 6131446
dc.identifier.doi https://doi.org/10.31274/rtd-180813-16498
dc.identifier.s3bucket isulib-bepress-aws-west
dc.identifier.submissionpath rtd/1924
dc.identifier.uri https://dr.lib.iastate.edu/handle/20.500.12876/73228
dc.language.iso en
dc.source.bitstream archive/lib.dr.iastate.edu/rtd/1924/r_3328089.pdf|||Fri Jan 14 21:53:50 UTC 2022
dc.subject.disciplines Analytical Chemistry
dc.subject.keywords Chemistry
dc.subject.keywords Analytical chemistry
dc.title Automated high throughput protein crystallization screening at nanoliter scale and protein structural study on lactate dehydrogenase
dc.type article
dc.type.genre dissertation
dspace.entity.type Publication
relation.isOrgUnitOfPublication 42864f6e-7a3d-4be3-8b5a-0ae3c3830a11
thesis.degree.level dissertation
thesis.degree.name Doctor of Philosophy
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