Defense Date

3-27-2025

Graduation Date

Spring 5-10-2025

Availability

One-year Embargo

Submission Type

dissertation

Degree Name

PhD

Department

Chemistry and Biochemistry

School

School of Science and Engineering

Committee Chair

Michael Cascio

Committee Member

Mihaela Rita Mihailescu

Committee Member

Michael Van Stipdonk

Committee Member

Joseph McCormick

Abstract

Cryomicroscopy, crystallography and NMR have been considered the “gold standard” of structural biophysics for some time. Recent successes in using these techniques has provided us with a plethora of insight into the architecture of different macromolecules like channels, transporters, and other molecular machines. However, information is often limited to a single allosteric state with the macromolecule in a non-native environment. Additionally, these traditional methods tend to be expensive and labor-intensive, requiring samples that are concentrated and highly purified. Chemical crosslinking coupled with mass spectrometry (CX-MS) has emerged as the ideal accessory for the aforementioned structural tools. In order to address these limitations, we will utilize CX-MS to study the lipid accessibility of the serotonin transporter (SERT) as a function of conformational state. In the absence of ligand, SERT is primarily in its outward-facing apo state. In the presence of ibogaine, a psychoactive alkaloid and allosteric modulator of SERT, the dynamic equilibrium that exists between SERT’s state-dependent conformation shifts toward an inward facing conformation. An X8C construct of rat SERT was purified from baculovirus infected cells and reconstituted in unilamellar lipid vesicles containing roughly 15% (mol/v) or higher cholesterol doped with azi-cholesterol, a non-specific, photoactivatable crosslinker. Comparative CXMS studies were then conducted in the absence or presence of ibogaine. Post photoactivation, crosslinked rSERT was digested and mass fingerprinted using an Agilent 6500 electrospray ionization quadrupole time-of-flight mass spectrometer (ESI-Q-TOF MS). Covalently modified peptides were identified and subjected to MSMS analysis to further refine the site of covalent attachment within the mass-shifted peptide. Our studies identified multiple regions of cholesterol interaction in both the apo and ibogaine-bound, inward-facing states. Additionally, our studies confirmed occupancy of cholesterol in a TM helicies 1, 5, 7 binding site predicted by computational studies of the apo state. Unique crosslinking patterns observed in this study, in complement with other lipid probing studies conducted in our lab, suggest the site for the SERT dimer interface for the first time. In essence, that absence of cholesterol binding to TM helices 10-12 suggest this region may be involved in SERT dimer formation. Similar cholesterol CX-MS studies were also used to identify sites of cholesterol interaction in protein tyrosine phosphatase type 1B (PTP1B). PTP1B is a regulator of insulin receptor signaling whose activity has recently been shown be regulated as a function of cholesterol concentration. Furthermore, when exposed to cholesterol for a prolonged period of time, PTP1B demonstrates some autoproteolytic activity producing a band shift visualized via SDS-PAGE. Purified PTP1B samples were exposed to endogenous cholesterol both in the presence and absence of photoactive azi-cholesterol, crosslinked, digested, and analyzed by MS fingerprinting. Our studies were able to disclose where both endogenous and photoactive cholesterol interacts with PTP1B and the region of autoproteolysis. We determined two distinct sites of interaction for endogenous cholesterol and azi-cholesterol. Endogenous cholesterol crosslinks PTP1B within a predicted cholesterol recognition amino acid consensus sequence (119L(KCAQ)Y(WPQ)K128). Azi-cholesterol instead, interacts with a hydrophobic cavity, “the VK cavity”. This demonstrates the selectivity PTP1B has for its potentially catalytic cholesterol binding site In addition, we observed that the C-terminus of the protein is autopreoteolyzed, consistent with observed gel shifts observed after cholesterol incubation.

Language

English

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Included in

Biochemistry Commons

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