Date of Award

12-2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Interdisciplinary Program

Track

Cancer and Developmental Biology

Research Advisor

Jian Zuo, Ph.D.

Committee

Linda M. Hendershot, Ph.D James I. Morgan, Ph.D Stephen White, DPhil Dianna A. Johnson, Ph.D

Abstract

The sensitivity of mammalian hearing relies upon the ability to amplify sound. Outer hair cells (OHCs) in the cochlea have motile properties that support this function. The motor protein called prestin resides in the lateral wall of the OHCs. Prestin undergoes voltage-dependent conformational changes, which correlates to the movement of charge through the membrane or non-linear capacitance (NLC). This property underlies OHC electromotility. A mechanistic understanding of prestin’s function remains unknown. Previous research has tied prestin’s C-terminus (residues 499-799) to the voltage-dependent events that occur in the membrane. The folding and biochemical properties of prestin’s C-terminus were characterized. This information was used to probe the function of the C-terminus.

Sequence analysis and limited proteolysis indicated that a 60-70 residue loop partitions the sulfate transporter and anti-sigma factor (STAS) domain in the C-terminus. To investigate the structural and functional role of the ‘loop’ (residues 571-635), the sequence was deleted (Δ) from prestin cDNA and transfected into HEK 293T cells. Whole-cell voltage clamp experiments confirmed that prestin Δ571-635 exhibited NLC; however, the voltage-capacitance curve had an increased slope-factor compared to wild type. This result suggested that the mutation affected the protein’s voltage sensor. The fact that the deletion protein exhibited NLC implies that the mutation did not adversely affect the structure. It was hypothesized that only a small subset of residues, attributed to the STAS domain, contribute to the structure in prestin’s C-terminus. To characterize the C-terminus structure, a series of mutant proteins were made with sequence deleted from the regions bordering the STAS domain. The implication being that the deletion would destabilize the protein if it disrupted important structure. On that premise, nuclear magnetic resonance spectroscopy (NMR), and analytical ultracentrifugation were used to characterize the folding and stability of the deletion mutants.

The results showed that the deletion mutants existed as folded, monomeric proteins in solution. Interestingly, several of the proteins displayed an ensemble of folding conformations that ranged from compact/globular to prolate/ellipsoid. Based on the deletions, the data suggests that the STAS domain consists of two subdomains (STAS A and STAS B) separated by a loop approximately 65 residues long. To understand the basis of the observed conformations, surface plasmon resonance (SPR) imaging determined whether binding occurs between the two subdomains. The data showed that the STAS A and B subdomains bind in a specific and reversible manner characterized by a slow on-rate with weak affinity. NMR data showed chemical shifts consistent with a folded protein; however, differences of peak intensity suggested that movement occurred within the structure of the C-terminus. The observations from the NMR and SPR studies appeared to be consistent with conformations observed from the analytical ultracentrifugation. In summary, the data suggests that the C-terminus contributes to the voltage sensor function. These behaviors of the protein in solution appear to be an innate feature of the C-terminus and may define the unique properties of prestin within the SLC26 protein family.

DOI

10.21007/etd.cghs.2009.0249

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