Semester of Graduation

Spring 2018

Degree

Master of Biological Science (MBioSci)

Department

Biological Sciences

Document Type

Thesis

Abstract

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.

Date

4-4-2018

Committee Chair

LiCata, Vincent

DOI

10.31390/gradschool_theses.4693

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