Design & engineering of conformationally switchable artificial metalloproteins (swArMs)

Metalloenzymes are nature’s exquisite biocatalysts harnessing the catalytic power of their embedded metallocofactors through the interplay of structural dynamics and electronic modulation. Conformational changes in metalloenzymes orchestrate the intricate choreography of substrate binding, active site reorganization, and product release, thereby modulating metallocofactor reactivity and enabling catalysis. However, understanding the mechanisms of reactions catalyzed by native metalloenzymes has been challenging due to the inherent complexity of these systems. Artificial metalloproteins (ArMs) have emerged as excellent models for native metalloenzymes, but most existing ArMs lack the conformational flexibility that is crucial for metalloenzyme function. This dissertation presents the design and engineering of conformationally switchable artificial metalloproteins (swArMs), to elucidate the critical interplay between protein dynamics and metallocofactor reactivity. Capitalizing on the allosterically regulated Escherichia coli glutamine-binding protein (GlnBP) as a model scaffold, we have developed swArMs that undergo large-scale structural rearrangements upon allosteric effector binding. This conformational transition modulates the microenvironment surrounding the installed metallocofactor, enabling the investigation of metallocofactor regulation driven by allostery. The first chapter discusses the site-specific installation of Co(dmgH)2(X) cofactors (dmgH = dimethylglyoxime, X = N3-, CH3-, iPr-) within GlnBP via direct Co–S cysteine ligation. Characterization via single-crystal X-ray diffraction and spectroscopic techniques such as fluorescence, circular dichroism, and infrared spectroscopy provide detailed insights into the architecture of the engineered swArM. Furthermore, our studies demonstrate that in swArMs containing the Co(dmgH)2(CH3) cofactor, the protein scaffold effectively stabilizes the Co–S bond. However, conformational changes induced by allosteric binding accelerate the cleavage of this bond, highlighting the swArM’s ability to regulate metallocofactor reactivity through conformational dynamics. Building on this foundation, the second chapter discusses a structure-informed computational approach to engineer swArMs with enhanced hydrogen-bonding (H-bonding) interactions surrounding the metallocofactor site upon conformational change. Molecular dynamics simulations were employed to identify key residues that impart glutamine-responsive control over the metallocofactor microenvironment, as probed experimentally by infrared spectroscopy. This rational design strategy not only recapitulates structural regulation in native metalloenzymes but also yields improved biological probes for future biosensing applications. The third chapter explores the incorporation of valence tautomeric cobalt-dioxolene [Co(diox)] and platinum(II) diimine complexes into the swArM scaffold. Strategies for bioconjugating these metallocofactors, either by assembling them within the protein matrix or through synthesis outside the protein and subsequent attachment to the protein, are investigated. While challenges were encountered, this chapter delineates design principles for successful metallocofactor incorporation, emphasizing the need for stable, soluble complexes with readily substitutable ligands compatible with the protein scaffold. Recognizing the potential of swArMs in cancer diagnostics and therapeutics, the final chapter focuses on bioconjugating gadolinium(III)-based magnetic resonance imaging (MRI) contrast agents to the GlnBP scaffold. By leveraging the upregulated glutamine uptake in cancerous cells, these glutamine-sensitive probes could enhance the sensitivity and specificity of MRI for cancer imaging and monitoring tumor progression and treatment response. Through the development of swArMs, this dissertation establishes a robust platform for systematically investigating the intricate relationships between conformational dynamics and metallocofactor regulation. Integrating spectroscopic techniques, structural characterization, and computational modeling could provide helpful insights into the mechanisms governing metalloprotein function. Ultimately, this work paves the way for the rational design of biocatalysts with tailored reactivity, while also advancing cancer diagnostics through the development of metabolite-responsive imaging probes.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2024

Author dc:creator

• Fatima, Saman

Contributors dc:contributor

• Olshansky, Lisa
• Murphy, Catherine
• Gruebele, Martin
• Manesis, Anastasia

• Metalloenzymes
• Bioinorganic
• Chemistry
• Metalloproteins
• Conformational
• Switchable
• Design
• Artificial
• Engineering
• Allosteric
• Metals
• Dynamics
• Hydrogen-bonding
• Mri Probes
• Glutamine

Statement dc:rights

• Copyright 2024 Saman Fatima

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/125708

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F125708