eCommons

 

DEVELOPMENT OF X-RAY TECHNIQUES AND STRUCTURAL CHARACTERIZATION OF OXYGEN ADAPTATION IN TWO IRON-SULFUR PROTEINS

Access Restricted

Access to this document is restricted. Some items have been embargoed at the request of the author, but will be made publicly available after the "No Access Until" date.

During the embargo period, you may request access to the item by clicking the link to the restricted file(s) and completing the request form. If we have contact information for a Cornell author, we will contact the author and request permission to provide access. If we do not have contact information for a Cornell author, or the author denies or does not respond to our inquiry, we will not be able to provide access. For more information, review our policies for restricted content.

No Access Until

2025-09-05
Permanent Link(s)

Other Titles

Abstract

Proteins containing iron-sulfur (FeS) clusters within facultative anaerobes, organisms that can transition between aerobic and anaerobic metabolism, can provide a window into the evolutionary transition of life before oxygenation of the planet to after. However, the experimental design necessary to study oxygen-sensitive FeS cluster containing proteins, is often nontrivial. Performing experiments to study detailed molecular mechanisms in the absence of oxygen requires significant and diligent effort. This thesis will focus on two FeS proteins from facultative anaerobes, Escherichia coli and Saccharomyces cerevisiae, and the development and application of advanced structural techniques, such as serial crystallography, small-angle X-ray scattering (SAXS), and cryo-electron microscopy (cryoEM). Towards room-temperature crystallography studies of metalloproteins, I describe the development of a fixed-target sample preparation and mounting system for serial synchrotron crystallography in collaboration with MiTeGen and the Cornell High Energy Synchrotron Source (CHESS). I then describe the development of anoxic SAXS at CHESS station ID7A and apply the system to perform the first structural characterization of the E. coli transcription factor, Fumarate and Nitrate Regulation (FNR) protein, and its use of oxygen-sensing [4Fe-4S] clusters. Finally, I describe a model of the S. cerevisiae diphthamide biosynthesis complex Dph123-EF2 obtained by cryoEM and AlphaFold Multimer that hints at the mechanism by which a catalytically essential [4Fe-4S] cluster is rescued in the presence of oxygen. Altogether, the advances described within this thesis have contributed to the field of structural biology by providing two new avenues of experimental techniques that can be utilized by researchers throughout the world to characterize samples that were previously very difficult if not impossible to characterize. The results presented in this thesis also provide insight into how facultative anaerobes may have overcome or taken advantage of the oxygen sensitivity of FeS clusters.

Journal / Series

Volume & Issue

Description

261 pages

Sponsorship

Date Issued

2023-08

Publisher

Keywords

Biochemistry; Biophysics; Chemical biology; Molecular biology

Location

Effective Date

Expiration Date

Sector

Employer

Union

Union Local

NAICS

Number of Workers

Committee Chair

Ando, Nozomi

Committee Co-Chair

Committee Member

Pollack, Lois
Lin, Hening

Degree Discipline

Chemistry and Chemical Biology

Degree Name

Ph. D., Chemistry and Chemical Biology

Degree Level

Doctor of Philosophy

Related Version

Related DOI

Related To

Related Part

Based on Related Item

Has Other Format(s)

Part of Related Item

Related To

Related Publication(s)

Link(s) to Related Publication(s)

References

Link(s) to Reference(s)

Previously Published As

Government Document

ISBN

ISMN

ISSN

Other Identifiers

Rights

Attribution 4.0 International

Types

dissertation or thesis

Accessibility Feature

Accessibility Hazard

Accessibility Summary

Link(s) to Catalog Record