Degree
Doctor of Philosophy (PhD)
Department
Biological Science
Document Type
Dissertation
Abstract
P. ingrahamii is a halo-psychrophilic bacterium isolated from Arctic sea ice. We have cloned and purified the large fragment of the cold-active DNA polymerase I from P. ingrahamii, named Klenpin. The objective of this project is to directly compare the thermodynamic stability of Klenpin, and the salt dependence of that stability, with Klenow and Klentaq; two homologous polymerases from a mesophile (E. coli) and a thermophile (Thermus aquaticus).
We first examined the effects of salts on the thermal stability (Tm) of Klenpin and Klenow across the Hofmeister series. Significantly different trends were observed on the melting temperature changes for Klenpin versus Klenow, even in chaotropic salts such as guanidine hydrochloride at low concentrations. Klenow responded to Hofmeister stabilizing salts and destabilizing salts as expected, while Klenpin was stabilized by all salts, even those that normally destabilize proteins.
We further examined the salt effect on the structure of Klenpin and Klenow using CD spectra, Trp fluorescence quenching, and dynamic light scattering. The results show that salt alters the structure of Klenpin to a more compact conformation, whereas no significant influence is observed in Klenow. Salt also alters the unfolding process of Klenpin. In chemical denaturation, an intermediate is observed in the presence of salt, whereas Klenpin folds following a two-state model without salt. An increase in free energy of unfolding of Klenpin is also seen upon the addition of NaCl, and this improvement is mainly from the additional unfolding transition introduced due to the intermediate.
We hypothesize that non-specific screening of unfavorable electrostatic interactions on the surface of Klenpin may be responsible for many of the observed salt effects and several computational comparisons were conducted to test this hypothesis. Unlike those typically found for archaebacterial halophilic proteins, no significant difference is observed in the comparison of amino acid preference between Klenpin and Klenow. But the electrostatic surface potential maps of Klenpin, Klenow, and Klentaq show larger clusters of acidic and basic residues on the Klenpin surface, supporting our hypothesis that salts modify Klenpin through the nonspecific screening of unfavorable electrostatic interactions on the surface.
Date
11-2-2020
Recommended Citation
Zhu, Xinji, "Salt Dependence of Thermodynamic Stability of a Cold-Active DNA Polymerase I Fragment" (2020). LSU Doctoral Dissertations. 5389.
https://repository.lsu.edu/gradschool_dissertations/5389
Committee Chair
LiCata, Vince
DOI
10.31390/gradschool_dissertations.5389