Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Chemistry

Language

English (en)

Date of Award

5-18-2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Joshua A Maurer

Abstract

In this dissertation, we discuss the relationship between structure and function within the mechanosensitive channel of small conductance: MscS) superfamily. Specifically, we explore the function of the bacterial cyclic nucleotide gated: bCNG) ion channels and the lipid interactions of MscS in the closed and open state. Our main goal was to identify similarities between MscS and bCNG channels and to understand the differences between the two channel families.

The bCNG channel family is a unique subset of the MscS superfamily. These channels are structurally composed of a channel domain homologous to MscS, a non-conserved linker domain, and a cyclic adenosine monophosphate: cAMP) binding domain. Several bCNG channels gate in response to cAMP alone, indicating that these channels function as ligand gated ion channels. bCNG channels are highly homologous to the pore lining helix and the upper vestibule domain of MscS suggesting that these channels should gate in response to mechanical tension. The majority of bCNG channels are unable to gate in response to mechanical tension however, upon the removal of the cAMP binding domain limited mechanosensation is restored to bCNG channels.

Some bacterial genomes are predicted to encode for multiple bCNG homologues. RT-PCR analysis of several different bacterial strains shows that the mRNA for these bCNG homologues is detected at similar levels. When two bCNG channels are heterologously expressed in E. coli, they form heteromultimeric channels. These results suggest that bCNG channels are likely to form heteromultimers in vivo.

E. coli MscS is a well studied mechanosensitive channel. Previous research has identified critical residues for channel function; these residues are located throughout the channel but are not predicted to interact with the lipid tails. As a mechanosensitive channel, we would expect that the amino acids in the transmembrane domains interact with the hydrophobic lipid tails to allow for gating in response to mechanical tension. To identify these residues, an all atom molecular dynamics simulation was conducted on a closed state model of MscS. The combination of this simulation and phenotypic data identified seven residues in the closed state that interact with the lipids and are essential for channel function. The identification of lipid interacting residues in the closed state of MscS suggests that similar interactions would be important for the open state. However, no lipid interacting residues were identified in the open state of MscS. The lack of lipid interactions suggests that the open state of MscS does not have essential lipid interactions. This has lead us to propose a new gating paradigm for MscS, the Jack-In-The-Box gating model.

This study of the relationship between structure and function within the MscS superfamily has given us a greater understanding of the molecular interactions needed for channel function. We have learned that lipid interactions are essential for gating in response to mechanical tension and that the appendage of the C-terminal cAMP binding domain inhibits mechanical gating of bCNG channels.

Comments

This work is not available online per the author’s request. For access information, please contact digital@wumail.wustl.edu or visit http://digital.wustl.edu/publish/etd-search.html.

Permanent URL: http://dx.doi.org/10.7936/K72J68X2

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