Purification and Kinetic Analysis of Rhodospirillum Centenum Lov-Kinase and Its Putative Response Regulator

Persistent Link:
http://hdl.handle.net/10150/245080
Title:
Purification and Kinetic Analysis of Rhodospirillum Centenum Lov-Kinase and Its Putative Response Regulator
Author:
Olson, Kenneth Tadashi
Issue Date:
May-2012
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
LOV histidine kinase (LOV-HK) from the purple photosynthetic bacterium Rhodospirillum centenum was expressed and characterized along with its putative response regulator (RR). LOV-HK and RR were expressed in E. coli and purified in a two-step process of nickel affinity chromatography followed by gel filtration chromatography. Mass spectrometry showed that both proteins were successfully expressed. The LOV-HK dark-state spectrum has broad peaks at 362nm and 450nm characteristic of the flavin mononucleotide (FMN) chromophore in a tight binding pocket. The light-state spectrum showed ~33% bleach at 450nm, which slowly returned to the dark state. The recovery of LOV-HK had a rate constant of 4.13 x 10⁻⁴ S⁻¹ with a half-life of 28.0min. The recovery of LOV-HK was also investigated in the presence of RR and showed a rate of 4.17x 10⁻⁴ S⁻¹ (half-life of 27.7min). These results do not conclusively demonstrate an interaction between the two proteins, however further study is warranted.
Type:
text; Electronic Thesis
Degree Name:
B.S.
Degree Level:
bachelors
Degree Program:
Honors College; Biochemistry and Molecular Biophysics
Degree Grantor:
University of Arizona

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titlePurification and Kinetic Analysis of Rhodospirillum Centenum Lov-Kinase and Its Putative Response Regulatoren_US
dc.creatorOlson, Kenneth Tadashien_US
dc.contributor.authorOlson, Kenneth Tadashien_US
dc.date.issued2012-05-
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractLOV histidine kinase (LOV-HK) from the purple photosynthetic bacterium Rhodospirillum centenum was expressed and characterized along with its putative response regulator (RR). LOV-HK and RR were expressed in E. coli and purified in a two-step process of nickel affinity chromatography followed by gel filtration chromatography. Mass spectrometry showed that both proteins were successfully expressed. The LOV-HK dark-state spectrum has broad peaks at 362nm and 450nm characteristic of the flavin mononucleotide (FMN) chromophore in a tight binding pocket. The light-state spectrum showed ~33% bleach at 450nm, which slowly returned to the dark state. The recovery of LOV-HK had a rate constant of 4.13 x 10⁻⁴ S⁻¹ with a half-life of 28.0min. The recovery of LOV-HK was also investigated in the presence of RR and showed a rate of 4.17x 10⁻⁴ S⁻¹ (half-life of 27.7min). These results do not conclusively demonstrate an interaction between the two proteins, however further study is warranted.en_US
dc.typetexten_US
dc.typeElectronic Thesisen_US
thesis.degree.nameB.S.en_US
thesis.degree.levelbachelorsen_US
thesis.degree.disciplineHonors Collegeen_US
thesis.degree.disciplineBiochemistry and Molecular Biophysicsen_US
thesis.degree.grantorUniversity of Arizonaen_US
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