Cloning, Purification, and Preliminary DNA-binding and Unfolding Results for the DNA Polymerase I from the Psychrophile Psychromonas ingrahamii
Psychromonas ingrahamii is a psychrophilic bacterium that lives in Arctic polar sea ice and grows at a temperature range of -12 to 10º C. This bacterium resides within veins inside the ice where the salinity is high, resulting in a freezing point depression and liquid water. The large fragment of D...
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ftlouisianastuir:oai:repository.lsu.edu:gradschool_theses-5694 2024-09-15T18:35:23+00:00 Cloning, Purification, and Preliminary DNA-binding and Unfolding Results for the DNA Polymerase I from the Psychrophile Psychromonas ingrahamii Baker, John Tod 2018-04-04T07:00:00Z application/pdf https://repository.lsu.edu/gradschool_theses/4693 https://doi.org/10.31390/gradschool_theses.4693 https://repository.lsu.edu/context/gradschool_theses/article/5694/viewcontent/Baker_thesis.pdf unknown LSU Scholarly Repository https://repository.lsu.edu/gradschool_theses/4693 doi:10.31390/gradschool_theses.4693 https://repository.lsu.edu/context/gradschool_theses/article/5694/viewcontent/Baker_thesis.pdf LSU Master's Theses Psychromonas ingrahamii Thermodynamics High Salt Concentration Gibbs-Helmholtz DNA Polymerase I Arctic Sea Ice Biochemistry Biophysics Molecular Biology text 2018 ftlouisianastuir https://doi.org/10.31390/gradschool_theses.4693 2024-08-08T04:27:14Z Psychromonas ingrahamii is a psychrophilic bacterium that lives in Arctic polar sea ice and grows at a temperature range of -12 to 10º C. This bacterium resides within veins inside the ice where the salinity is high, resulting in a freezing point depression and liquid water. The large fragment of DNA polymerase I from Psychromonas ingrahamii, called Klenpin, has been cloned, expressed, and purified in our laboratory. Although enzyme kinetic studies have been performed on a few psychrophilic enzymes, the thermodynamics of ligand binding and of protein stability have not been well studied for this class of extremophilic proteins. Here we present initial results on the salt dependence and temperature dependence of DNA-binding for Klenpin as well as unfolding results obtained for this protein. The KCl dependence shows that DNA-binding affinity decreases as the salt concentration is increased from 10 mM to 1 M. Salt addition titrations with KCl show that salt displaces the polymerase from the DNA up to approximately 0.5M KCl, after which increased [KCl] induces rebinding of the polymerase to the DNA. Rebinding of the polymerase to DNA continues to increase up to 4.3 M KCl. Such rebinding to DNA at high salt has previously been documented in glutamate salts for several polymerases, but rebinding in KCl has not been previously observed for any polymerase. Temperature dependence shows Klenpin binds to DNA with submicromolar affinity at 8º C but binding affinity increases as the temperature is raised to 25º C. Unfolding results show that under similar solution conditions Klenpin has a similar Tm of 62.7ºC to its mesophilic homolog from E. coli, Klenow. This Tm is slightly higher than that for Klenow. Text Sea ice LSU Digital Commons (Louisiana State University) |
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Open Polar |
collection |
LSU Digital Commons (Louisiana State University) |
op_collection_id |
ftlouisianastuir |
language |
unknown |
topic |
Psychromonas ingrahamii Thermodynamics High Salt Concentration Gibbs-Helmholtz DNA Polymerase I Arctic Sea Ice Biochemistry Biophysics Molecular Biology |
spellingShingle |
Psychromonas ingrahamii Thermodynamics High Salt Concentration Gibbs-Helmholtz DNA Polymerase I Arctic Sea Ice Biochemistry Biophysics Molecular Biology Baker, John Tod Cloning, Purification, and Preliminary DNA-binding and Unfolding Results for the DNA Polymerase I from the Psychrophile Psychromonas ingrahamii |
topic_facet |
Psychromonas ingrahamii Thermodynamics High Salt Concentration Gibbs-Helmholtz DNA Polymerase I Arctic Sea Ice Biochemistry Biophysics Molecular Biology |
description |
Psychromonas ingrahamii is a psychrophilic bacterium that lives in Arctic polar sea ice and grows at a temperature range of -12 to 10º C. This bacterium resides within veins inside the ice where the salinity is high, resulting in a freezing point depression and liquid water. The large fragment of DNA polymerase I from Psychromonas ingrahamii, called Klenpin, has been cloned, expressed, and purified in our laboratory. Although enzyme kinetic studies have been performed on a few psychrophilic enzymes, the thermodynamics of ligand binding and of protein stability have not been well studied for this class of extremophilic proteins. Here we present initial results on the salt dependence and temperature dependence of DNA-binding for Klenpin as well as unfolding results obtained for this protein. The KCl dependence shows that DNA-binding affinity decreases as the salt concentration is increased from 10 mM to 1 M. Salt addition titrations with KCl show that salt displaces the polymerase from the DNA up to approximately 0.5M KCl, after which increased [KCl] induces rebinding of the polymerase to the DNA. Rebinding of the polymerase to DNA continues to increase up to 4.3 M KCl. Such rebinding to DNA at high salt has previously been documented in glutamate salts for several polymerases, but rebinding in KCl has not been previously observed for any polymerase. Temperature dependence shows Klenpin binds to DNA with submicromolar affinity at 8º C but binding affinity increases as the temperature is raised to 25º C. Unfolding results show that under similar solution conditions Klenpin has a similar Tm of 62.7ºC to its mesophilic homolog from E. coli, Klenow. This Tm is slightly higher than that for Klenow. |
format |
Text |
author |
Baker, John Tod |
author_facet |
Baker, John Tod |
author_sort |
Baker, John Tod |
title |
Cloning, Purification, and Preliminary DNA-binding and Unfolding Results for the DNA Polymerase I from the Psychrophile Psychromonas ingrahamii |
title_short |
Cloning, Purification, and Preliminary DNA-binding and Unfolding Results for the DNA Polymerase I from the Psychrophile Psychromonas ingrahamii |
title_full |
Cloning, Purification, and Preliminary DNA-binding and Unfolding Results for the DNA Polymerase I from the Psychrophile Psychromonas ingrahamii |
title_fullStr |
Cloning, Purification, and Preliminary DNA-binding and Unfolding Results for the DNA Polymerase I from the Psychrophile Psychromonas ingrahamii |
title_full_unstemmed |
Cloning, Purification, and Preliminary DNA-binding and Unfolding Results for the DNA Polymerase I from the Psychrophile Psychromonas ingrahamii |
title_sort |
cloning, purification, and preliminary dna-binding and unfolding results for the dna polymerase i from the psychrophile psychromonas ingrahamii |
publisher |
LSU Scholarly Repository |
publishDate |
2018 |
url |
https://repository.lsu.edu/gradschool_theses/4693 https://doi.org/10.31390/gradschool_theses.4693 https://repository.lsu.edu/context/gradschool_theses/article/5694/viewcontent/Baker_thesis.pdf |
genre |
Sea ice |
genre_facet |
Sea ice |
op_source |
LSU Master's Theses |
op_relation |
https://repository.lsu.edu/gradschool_theses/4693 doi:10.31390/gradschool_theses.4693 https://repository.lsu.edu/context/gradschool_theses/article/5694/viewcontent/Baker_thesis.pdf |
op_doi |
https://doi.org/10.31390/gradschool_theses.4693 |
_version_ |
1810478534446022656 |