Date of Graduation

5-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Physics (PhD)

Degree Level

Graduate

Department

Physics

Advisor

Gregory J. Salamo

Committee Member

William F. Oliver III

Second Committee Member

Laurent Bellaiche

Third Committee Member

Ralph Henry

Fourth Committee Member

Woodrow Shew

Fifth Committee Member

Daniel Fologea

Abstract

Lysenin is classified as a pore-forming toxin protein that is isolated from the earthworm Eisenia fetida and consists of 297 amino acids [1]. Lysenin inserts large conducting pores (3.0-4.7 nm in diameter) into artificial membranes (BLM) which include sphingomyelin. These pores (channels) are open and oriented upon insertion into the bilayer lipid membrane. Lysenin channels gate at positive voltages (voltage-induced gating), but not at negative voltages. Lysenin pores also exhibit activity modulation in response to changes in ionic strength and pH, indicating that electrostatic interaction is responsible for Lysenin conductance activities. In this line of inquiries, and by modulating Lysenin electrostatic interactions, it was hypothesized that the electrical properties of Lysenin pores (channels) could be influenced by multivalent ions.

The macroscopic conductance of Lysenin channels was inhibited by addition of multivalent ions. The inhibition was concentration dependent and reversible by addition of chelating or precipitating agents. The ability of the examined multivalent ions to inhibit pore conductance depended on ionic charge and size. Taken together, these results indicate that the Lysenin channel has a binding site that is placed inside the channel and interacts electrostatically with multivalent ions resulting in a conductance response related to ionic number and size. The high sensitivity of Lysenin pores toward trivalent ions indicates that Lysenin channels could be used to develop novel biosensors for multivalent ion detection in environmental samples.

The dynamic interaction of Lysenin with multivalent ions was modeled based on the conductivity of the bulk solution and the status of Lysenin channels. The purpose of the model was to provide a mechanistic understanding of Lysenin gating. Using the experimental data, an equilibrium rate constant of the interaction between Lysenin and each multivalent ion was estimated. Each rate constant was related to the binding affinity of each ion with the binding site.

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