All ETDs from UAB

Advisory Committee Chair

Christie G Brouillette

Advisory Committee Members

Wayne J Brouillette

Champion C S Deivanayagam

Aaron L Lucius

John W Shriver

Document Type

Dissertation

Date of Award

2015

Degree Name by School

Doctor of Philosophy (PhD) College of Arts and Sciences

Abstract

PART I. Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) cause the lethal genetic disease cystic fibrosis (CF). The most common CF-causing mutation is the deletion of F508 (F508del) in its N-terminal nucleotide binding domain (NBD1), which causes CFTR misfolding and premature degradation. By studying the in vitro thermal unfolding of the isolated NBD1 ± F508del using differential scanning calorimetry (DSC), we show that F508del significantly reduces the thermodynamic and kinetic stability of NBD1. We propose that the non-native contact between the partially denatured F508del-NBD1 and other domains may cause the misfolding of F508del-CFTR. This work has led to the discovery that restoring NBD1 thermodynamic stability is necessary and sufficient to correct the F508del defect [He et al. 2005.]. In order to provide a practical guideline for the selection of detergents to use for CFTR extraction and purification, we studied the effect of detergents on the stability and folding of NBD1 by DSC and circular dichroism (CD). We show that several commonly used anionic detergents for CFTR purification denature NBD1 and compromise the stability and function of the full-length CFTR. In general, all detergents destabilize NBD1 and the detergent effect on NBD1 can predict their effect on CFTR during purification to some degree. We then investigated the thermal unfolding of P-glycoprotein (Pgp), a prototypical ABC exporter that has been used as a model for CFTR, and its NBDs by DSC, CD and fluorescence spectroscopy. Pgp unfolds in two distinct steps, suggesting the presence of two relatively independent cooperative unfolding units. By comparing the unfolding of Pgp with that of the isolated NBDs in the absence and presence of MgATP, we propose that each cooperative domain contains one NBD and two intracellular loops. This assignment is consistent with the available Pgp structures that show the NBDs completely dissociated. Energetic coupling between the NBDs is observed for a catalytic inactive mutant, E552A/E1197A, upon binding of MgATP, which suggests the formation of the NBD1-NBD2 heterodimer. We conclude that thermal unfolding studies can provide insights into the dynamics of multi-domain membrane proteins that crystal structures may not be able to provide. PART II. NAD+ synthetase is a 60-kDa homodimeric enzyme that contains an extensive dimer interface that is necessary for its function. In the presence of relatively low concentrations of Mg2+, K+ and NH4+, the protein unfolds via a two-state mechanism with simultaneous dissociation of the dimer (N2 ⇄ 2U), indicating high energetic coupling between the two monomers. 2.5% (v/v) DMSO reduces the association constant of the monomers, and induces the formation of a monomeric intermediate in the unfolding pathway [Yang ZW, Tendian SW, Carson WM, Brouillette WJ, Delucas LJ, Brouillette CG. Protein Sci. 2004. 13(3):830-41.]. In the present study, we have discovered that removal of Mg2+, but not K+ or NH4+, from the buffer also causes a change in the unfolding mechanism of the homodimer. The unfolding of NADS in salt-free buffer is best described by a three-state model with a dimeric intermediate (N2 ⇄ I2 ⇄ 2U). The unfolding mechanism is further modulated by the presence of DMSO, and the effects of DMSO on the two transitions are different. It appears that Mg2+ and DMSO affect different interfaces within the dimer. Based on the structural changes accompanying the unfolding and the changes in unfolding cooperativity, we propose that NADS homodimer is composed of two energetic domains of different intrinsic stability. The dividing line between the energetic domains is along the two equivalent substrate binding clefts which are perpendicular to the dimer interface. This domain assignment effectively divides the protein into four quadrants which are separated by the dimer interface and the energetic domain interface. Mg2+ increases the structural cooperativity of the NADS dimer by energetically linking the two domains together. We show that the effect of Mg2+ is most likely due to screening of the electrostatic repulsion between the two energetic domains. The lowered structural cooperativity of NADS in bacterial cells may contribute to the low antibacterial activity of some classes of inhibitors identified from the cell-free enzymatic assay in which they bind to the conformation with high structural cooperativity.

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