University of Illinois Urbana-Champaign

Design & engineering of conformationally switchable artificial metalloproteins (swArMs)

Fatima, Saman

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

Description

Title
Design & engineering of conformationally switchable artificial metalloproteins (swArMs)
Author(s)
Fatima, Saman
Issue Date
2024-07-12
Director of Research (if dissertation) or Advisor (if thesis)
Olshansky, Lisa
Doctoral Committee Chair(s)
Olshansky, Lisa
Committee Member(s)
  • Murphy, Catherine
  • Gruebele, Martin
  • Manesis, Anastasia
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
  • metalloenzymes
  • bioinorganic
  • chemistry
  • metalloproteins
  • conformational
  • switchable
  • design
  • artificial
  • engineering
  • allosteric
  • metals
  • dynamics
  • hydrogen-bonding
  • MRI probes
  • glutamine
Abstract
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.
Graduation Semester
2024-08
Type of Resource
Thesis
Handle URL
https://hdl.handle.net/2142/125708
Copyright and License Information
Copyright 2024 Saman Fatima

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