ORCID
0000-0002-8995-3507
Date of Award
4-30-2024
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Many ion channels are multi-subunit complexes where oligomerization is an obligatory requirement for function as the binding axis forms the charged permeation pathway. However, the mechanisms of assembly within the membrane are largely unknown. Are ion channel complexes thermodynamically stable, and do they participate in dynamic equilibrium oligomerization reactions in membranes? If so, what are the molecular factors governing these reactions? In this thesis, I present several key advances in the study of ion channel oligomerization in membranes through quantitative studies of the dimerization equilibrium reaction of the inverted topology, homodimeric fluoride channel Fluc in lipid bilayers. Chapter 1 provides an introduction into the membrane protein folding problem as a multi-step process that occurs in the lipid bilayer which is a very different solvent than water. I discuss folding models that can help provide a conceptual framework of membrane protein structure formation and highlight the need to extend these models towards subunit oligomerization which is often an obligatory requirement for functional assembly. I review what fundamental insights into the driving forces of membrane protein oligomerization have been gained by previous thermodynamic analyses of membrane protein oligomerization and finally introduce the fluoride channel Fluc as a model system to study ion channel assembly in membranes. Chapter 2 describes the main methodologies applied in this thesis research, introducing the single-molecule photobleaching subunit capture approach, bulk FRET measurements, and functional analysis such as the liposomal fluoride flux assay and single-channel recordings. Next in Chapter 3, I report on a general methodology to obtain quantitative fluorophore labeling of oligomeric membrane proteins for single-molecule photobleaching studies to measure stoichiometry and reactive oligomerization in membranes. Single-molecule photobleaching analysis is a useful approach to quantify the oligomerization reactions in membranes as it provides a binary readout of a fluorophore attached to a protein subunit at dilute conditions. However, any quantification of stoichiometry also critically requires knowing the probability that a subunit is fluorescently labeled. Thus, I describe cysteine site screening on two model systems, CLC-ec1 and Fluc by providing a rationale for building a common-sense list of candidate cysteine sites, followed by rigorous testing of accessibility using the Ellman’s assay, Cy5-maleimide labeling, functional assays of the labelled protein and single-molecule total internal reflection microscopy imaging. I find that initial, well rationalized choices are not strong predictors of success, as rigorous testing of the labeling sites shows that only ~ 30% of sites end up being useful for single-molecule photobleaching studies. Chapter 4 presents an approach to estimate labeling yields using the photobleaching probability distribution of an intrinsic dimeric control and determine the dissociation constant of reactive dimerization constructs without prior knowledge of the fluorophore labeling yield. I find that iterative fitting of an experimental dimeric photobleaching probability distribution of CLC-ec1 to an expected dimer model allows to estimate the fluorophore labeling yields and find agreement with direct measurements of labeling of the purified protein by UV-VIS absorbance before reconstitution. Using this labeling prediction, similar estimation methods are applied to then determine the dissociation constant of reactive dimerization constructs of CLC-ec1 without prior knowledge of the labeling yield. In Chapter 5, I apply these newly developed tools to investigate the dimerization reaction of Fluc in membranes. While the wild-type channel is a long-lived dimer, I leverage a known mutation, N43S, that weakens Na+ binding in a buried site at the interface, thereby unlocking the complex for reversible association in lipid bilayers. Single-channel recordings show that Na+ binding is required for fluoride conduction while single-molecule microscopy experiments demonstrate that N43S Fluc exists in a dynamic monomer-dimer equilibrium in the membrane, even following removal of Na+. Quantifying the thermodynamic stability while titrating Na+ indicates that dimerization occurs first, providing a membrane-embedded binding site where Na+ binding weakly stabilizes the complex. To understand how these subunits form stable assemblies while presenting charged surfaces to the membrane, molecular dynamics simulations were carried out, which show the formation of a thinned membrane defect around the exposed dimerization interface. In simulations where subunits are permitted to encounter each other while preventing protein contacts, spontaneous and selective association at the native interface is observed, where stability is achieved by mitigation of the membrane defect. These results suggest a model wherein membrane-associated forces drive channel assembly in the native orientation while subsequent factors, such as Na+ binding, result in channel activation. Then in Chapter 6, to understand the protein dependent factors that define this reaction, I demonstrate several strategies for perturbing dimerization by investigating how mutations of interface-lining residues affect dimerization stability, channel activity or both. Site-directed mutagenesis was employed following four strategies: (1) removal of non-polar interactions at the interface; (2) mutation of glycine residues in TM helix 1; (3) removal of potential polar side chain interaction by threonine-to-valine substitutions; (4) and introduction of bulky tryptophan residues at the interface. Using single-molecule photobleaching analysis to investigate dimer stability in membranes, bulk FRET measurements to probe the dimerization kinetics, and single channel bilayer recordings to test protein function, I find that two constructs, G15A/G19A (GAGA) and A101W, demonstrate dynamic dimerization behavior upon the WT Fluc background. While both mutants show changes in dimerization kinetics and Na+ affinity, they show little effects on thermodynamic stability and still support a two-step equilibrium dimerization reaction as observed for N43S Fluc. Finally, in Chapter 7 I discuss how the studies on the fluoride channel Fluc presented in this thesis contribute to our understanding of ion channel assembly more broadly as dynamic assembly may be generalizable to other ion channels. I highlight that dynamic assembly provides an additional mechanism for modulating protein activity and discuss potential consequences of disruption of the thermodynamic balance of assembled and disassembled states for example through mutations. Further, I discuss how disassembled states may remain hidden in many of our current methods to study ion channels and provide a roadmap for future studies that make use of methodology and strategies that allow to observe these previously hidden states. I further discuss how the results presented here, along with previous studies, support the membrane as providing a generalizable driving force for specific, oriented membrane protein assembly in membranes. This provides a possibility that non-specific lipid association may modulate membrane protein equilibrium via the mechanism of preferential solvation. Finally, I discuss how the dimerization mechanism and Na+ binding of the dual-topology homodimer Fluc presents a model for inverted-topology Na+-coupled transporter folding and Na+-coupled substrate transport.
Language
English (en)
Chair and Committee
Janice Robertson
Recommended Citation
Ernst, Melanie, "Dimerization of the Fluoride Channel Fluc in Membranes" (2024). Arts & Sciences Electronic Theses and Dissertations. 3019.
https://openscholarship.wustl.edu/art_sci_etds/3019