ORCID
https://orcid.org/0000-0003-4883-4744
Date of Award
12-2023
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
The efforts of this thesis are directed towards understanding the thermodynamics and kinetics underlying the dimerization reaction of a chloride/proton antiporter membrane protein, CLC-ec1, in lipid membranes. Due to the inherent challenges of working with membrane proteins, thermodynamic and kinetic information for the protein association reactions in membranes has been historically limited. The research presented in this thesis demonstrates the technological advancements for these measurements in a model membrane protein system, thereby setting an important milestone for the field. Since CLC-ec1 dimerizes via a greasy, membrane embedded binding interface, the physical driving forces involved in this reaction were unknown. It further presents a thermodynamic dissection of CLC-ec1 dimerization in lipid bilayers by carrying out a temperature-dependent van ’t Hoff analysis using the single-molecule photobleaching subunit-capture approach. This revealed a large negative change in the heat capacity, ΔCP, in CLC-ec1 dimerization, representing the same thermodynamic signature as the hydrophobic effect. This investigation also led to the development of a method for measuring protein subunit-exchange in membranes by bulk Förster resonance energy transfer (FRET), showing slow dissociation kinetics at ambient temperatures. We found that the subunit-exchange speeds up with temperature, following an Arrhenius relationship and suggesting that there is a high transition state energy barrier to dissociation. This work led to the hypothesis that the energy barrier is formed by states along the dissociation pathway where the subunits separate but are incompletely solvated, introducing cavity volumes. To investigate this hypothesis, coarse grained molecular dynamics simulations were set up for CLC-ec1 subunits of different separations using MARTINI force field to characterize cavity formation. The simulations were repeated with mixed lipid bilayers containing smaller molecules, such as lysolipids and benzene to see if these solvents can reduce the cavity volumes, with results showing that smaller size molecules at higher concentrations increase the overall packing density in the cavity between the CLC-ec1 subunits. Finally, since it is unclear as to the reason why CLC-ec1 evolved as a stable homodimer, an investigation was carried out into whether the dimerization reaction was linked to transport mechanism. Subunit-exchange experiments were carried out as a function of pH, a known environmental regulator of CLC-ec1 transport function, showing that at pH > 6.5, dissociation no longer occurs corresponding to conditions where CLC-ec1 transport function is reduced. In addition, the connection between transport mechanism and dimerization was examined by studying a channelized version of CLC- ec1 with binding site mutations E148A/Y445S. Our findings demonstrate that CLC dimerization is strongly linked to pH, but the dimerization is not closely linked to the transport mechanism. Altogether, the results of this research present an in-depth thermodynamic analysis of membrane protein dimerization in membranes and establish a foundation for future studies to understand membrane protein complex stability, kinetics, and regulation. Chapter 1 of this thesis presents an overview of previous research on CLC-ec1 as well as the study of membrane protein association equilibrium in membranes. Chapter 2 describes the main methodologies applied or developed in this thesis research, including the single-molecule photobleaching subunit capture approach, bulk FRET measurements, and chloride transport efflux assays. Chapter 3 presents a tutorial protocol of the Lambda III single-molecule total internal reflection fluorescence (TIRF) microscope, a pivotal piece of equipment used in investigating the thermodynamic stability of CLC-ec1 in membranes. It provides a step-by-step guide on alignment of the microscope, starting from the excitation pathway, emission pathway, and finally the alignment of the micromirrors for total internal reflection. The protocol also describes the basic physical principles of this TIRF microscope. Chapter 4 describes the thermodynamic dissection of CLC-ec1 dimerization equilibrium in lipid bilayers by performing a temperature dependent van ’t Hoff of the free energy of CLC-ec1 dimerization in two different lipid compositions. In addition, it presents a new methodology for measuring dynamic subunit-exchange in membranes by bulk FRET, revealing the remarkably slow kinetic behavior of this dynamic equilibrium reaction at ambient temperatures. Chapter 5 presents a computational modeling investigation of potential dissociation states of CLC-ec1 and whether there are cavities formed that are inaccessible to phospholipid solvent molecules between the CLC-ec1 subunits. The analysis then extends to the study of mixed membranes containing lysolipids and benzene to investigate whether these small molecules increase lipid solvation to provide rationale for lipid dependency of subunit dissociation kinetics. This analysis serves as the initial steps towards identifying a relationship between lipid solvation and the dimerization kinetics. Chapter 6 reports on an investigation into whether dimerization is linked to the regulation of functional mechanisms of CLC-ec1. To test this, we investigate the dimerization of wild type (WT) CLC-ec1 at different pH, which is known to regulate transport function, and examine the thermodynamic and kinetic stability of transporter vs. channelized versions of the protein. Finally, Chapter 7 provides a discussion of the impact of this research on understanding the kinetics and thermodynamics of membrane protein oligomerization in general with a focus on future directions that will expand on this foundational research.
Language
English (en)
Chair and Committee
Janice L Robertson, Anders E Carlsson
Committee Members
Baron Chanda, Shankar Mukherji, Ralf Wessel
Recommended Citation
Lee, Taeho, "Thermodynamics and Kinetics Analyses of CLC-ec1 Dimerization Reaction in Lipid Bilayers" (2023). Arts & Sciences Electronic Theses and Dissertations. 3110.
https://openscholarship.wustl.edu/art_sci_etds/3110