Date of Award

May 2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Physics

First Advisor

Valerică Raicu

Committee Members

Julie Oliver, David Frick, Ionel Popa, Peter Schwander

Keywords

Fluorescence, Förster resonance energy transfer, Oligomers, Quaternary structure, Receptors, Spectroscopy

Abstract

Proteins are of high interest in biophysics research due to the important roles they play within cells, such as sensing of chemical (ions and small molecules) and physical (e.g., light) stimuli, providing structure, transporting ions/molecules, signaling, and intercellular communication. The studies described in this dissertation focus on a particular type of membrane proteins known as G protein-coupled receptors (GPCR), which play a key role in cellular response to external stimuli. We used the sterile 2 α-factor mating pheromone receptor (Ste2), a prototypical class D GPCR present within Saccharomyces cerevisiae (baker’s yeast). Ste2 is responsible for initiating the second messenger signal transduction cascade present within a-type haploid yeast cells by binding to their natural agonist, α-factor, which is secreted by cells of the opposing mating cell type (α-type haploid). Ultimately, Ste2 signals the a-type haploid to grow a projection towards the gradient of secreted pheromone from α-type haploids to locate a mate and form a diploid cell and eventually reproduce through meiosis. The propensity of this receptor to form oligomeric complexes has been documented in several publications, though the size of the oligomer in terms of the number of subunits (or protomers), the geometry of the oligomer, as well as any changes in the oligomer size and geometry due to agonist ligand binding has been lacking. We studied oligomerization of the Ste2 receptors in living cells by means of Fluorescence (or Förster) Resonance Energy Transfer (FRET) utilizing variants of the green fluorescent protein (GFP) as fluorescent markers and two-photon fluorescence micro-spectroscopy for acquiring spectrally resolved fluorescence images of living cells. From the imaged cells, we calculate pixel-level distributions of apparent FRET efficiencies (termed Eapp histograms or FRET spectrograms), which contain “fingerprints” of the most abundant quaternary structure present in the form of distinct peaks. The relation between the number of peaks in an Eapp histogram and their positions along the distribution corresponds to the number and relative distances between protomers within an oligomer. By distilling the Eapp histogram peaks, we can calculate a meta-spectrogram (or meta-histogram) which contains the most probable quaternary structure measured across many cells which can then be modeled using a suitable oligomeric complex. This method is known as FRET spectrometry and using it we determined the quaternary structure status and stoichiometry of Ste2 homo-oligomers. In our pursuit to refine the precision of our analysis, we further determined that the quaternary structure of Ste2 changes geometry from absence to presence of α-factor pheromone. Quite satisfyingly, were also able to observe, for the first time in living cells, that introduction of ligand modulates the probability of occurrence of different quaternary structure sub-states of Ste2 oligomers. This finding provides important insights into the mode of action of hormonal factors in eukaryotic cells and may provide critical information which relates quaternary structure to function. In a second part of our study, we made an unforeseen observation that fluorescently labeled α-factor pheromone could traverse the plasma membrane of yeast cells into the cytosol and bind to internal membrane structures. Using fluorescence micro-spectroscopy and fluorescently labeled α-factor pheromone, we determined that this ligand is internalized in greater quantities by cells that display higher quantities of the age marker lipofuscin. We then tried to inhibit the penetration of α-factor through the yeast membranes to understand the mechanism of action. We altered the environmental pH of the cells to test prior knowledge that neutral pH can induce the inner leaflet of the plasma membrane to acquire a net negative charge through deprotonation of multiple types of phospholipid species residing in the inner leaflet. This leaves the positive charge of the α-factor lysine virtually unaffected, which in turn generates a driving force towards the interior of the cell. We observed a reduction in ligand internalization by at more acidic pH, though it was not inhibited entirely. We then turned to molecular dynamics simulations for a model of the yeast membrane in the presence of increasing numbers of ligand molecules. Our findings show that, when a critical number of α-factor molecules are present, and embed themselves into the membrane, a porous like structure is formed, which may explain the ability of α-factor pheromone to act as a cell penetrating peptide. Overall, our study indicates that, while through binding to its cognate receptor (Ste2) the ligand triggers a set of changes in the quaternary (and tertiary) structure of Ste2 leading to an elaborate response of the cell for locating its mating partner, it may also trigger cellular responses in the absence of cognate receptor via penetration through the plasma membrane and its detection inside the cell. The role of the latter process may very well be that of stimulating the cell to produce receptor that is then delivered to the plasma membrane to help the cell identify the location of its mating partner via the former process.

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