Theses and Dissertations

Issuing Body

Mississippi State University

Advisor

Fitzkee, Nicholas C.

Committee Member

Lewis, Edwin A.

Committee Member

Emerson, Joseph P.

Committee Member

Mlsna, Todd E.

Committee Member

Mlsna, Debra Ann

Date of Degree

12-1-2017

Original embargo terms

MSU Only Indefinitely

Document Type

Dissertation - Campus Access Only

Major

Chemistry

Degree Name

Doctor of Philosophy

College

College of Arts and Sciences

Department

Department of Chemistry

Abstract

Calcineurin (CaN) plays an important role in T-cell activation, cardiac system development, and nervous system function. Previous studies have suggested that the regulatory domain (RD) of CaN binds Calmodulin (CaM) towards the N-terminal end of CaN. Calcium-loaded CaM activates the serine/threonine phosphatase activity of CaN by binding to the regulatory domain, although the mechanistic details of this interaction remain unclear. It is thought that CaM binding at the RD displaces the auto inhibitory domain (AID) from the active site of CaN, which activates phosphatase activity. In the absence of calcium-loaded CaM, the RD is at least partially disordered, and binding of CaM induces folding in the RD. Previous studies have shown that an ?-helical structure forms in the N-terminal half of the RD, but organization may occur in the C-terminal region as well. Here, we are presenting a model for the structural transition of the full length RD as it binds to CaM. Using nuclear magnetic resonance (NMR) spectroscopy, we have successfully assigned >85% of the 15N, 13C?, 13C? and HN chemical shifts of the unbound, regulatory domain of CaN. Secondary chemical shifts support a model where the RD is highly disordered. Our study of the CaM and CaN interaction supports the formation of a distal helix in the region between the AID and calmodulin-binding region. Heat capacity changes upon binding predict that 43 residues fold when CaM binds to CaN, consistent with the formation of this distal helix. Paramagnetic relaxation enhancement (PRE) studies of this interaction suggest a potential binding mode where the distal helix binds to CaM near residues I10-A11. Mutagenesis in the distal helix disrupts PREs, further supporting this hypothesis. Together, these data suggest that the interactions between CaM and the distal helix of CaN can be important in regulation of phosphatase activity.

URI

https://hdl.handle.net/11668/18716

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