Thesis: Ph. D. in Biological Chemistry, Massachusetts Institute of Technology, Department of Chemistry, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Ribonucleotide reductase (RNR) catalyzes the reduction of nucleotides to 2'- deoxynucleotides. The focus of this thesis is the F coli class la RNR, which is comprised of two homodimeric subunits, [alpha]2 and [beta]2, forming an active [alpha]2[beta]2 complex. The [beta]2 subunit harbors the stable diferric-tyrosyl radical cofactor (Y 122*) that reversibly oxidizes the active site cysteine (C₄₃₉) in [alpha]2. This oxidation requires a long-range radical transport (RT) pathway consisting of proton-coupled electron transfer (PCET) events through redox-active aromatic amino acid residues: Y₁₂₂* <--> [W₄₈] <--> Y₃₅₆ in [beta]2 to Y₇₃₁ <--> Y₇₃₀ <--> C₄₃₉ in [alpha]2. Once formed, the transient C₄₃₉* initiates nucleotide reduction. Both the long-range oxidation and the nucleotide reduction chemistries are kinetically masked by rate-limiting protein conformational change(s). To overcome this conformational change, the unnatural amino acid probe 3-aminotyrosine (NH₂Y) has been sitespecifically incorporated at multiple positions (Y₃₅₆, Y₇₃₁, Y₇₃₀) into the RT pathway. Herein, the NH₂Y probe is characterized as pertaining to the previously demonstrated ability for NH₂Y-incorporated RNR (NH₂Y-RNR) to form product. The reduction potential of NH₂Y produces a thermodynamic barrier that RNR cannot overcome. To explain NH₂Y-RNR activity, mass spectrometry was used for relative quantitation of contaminating wt-RNR in the NH₂Y-RNR, lending credence to the fact that the NH₂Y-RNRs are actually inactive. These results provide clarity to the long-standing mystery behind the low activities of the NH₂Y-RNRs. The use of the NH₂Y probe to generate stable radicals on the RT pathway has revealed further remarkable insight, demonstrating a hydrogen bonding network in the [alpha]2 subunit by employing advanced EPR methods on NH₂Y₇₃₀* and NH₂Y₇₃₁*. The evidence for a collinear PCET mechanism is provided with the NH₂Y₇₃₀/Y₇₃₁F and NH₂Y₇₃₁/C₄₃₉A mutants. Mutation of an R₄₁₁ to alanine in [alpha]2 allowed the detection of a "flipped" NH₂Y₇₃₁* conformation using advanced EPR techniques. Herein, photo cross-linked RNR is studied by tandem mass spectrometry (MS/MS). The study of a photo cross-linked [alpha]2[beta]2 complex using a 4-N-maleimido-benzophenone covalently attached to the C-terminal tail of [beta]2 yielded no photo cross-linked peptides. These studies taken together provide additional insight at the [alpha][beta] interface and provide additional tools to study this interaction. / by Wankyu Lee. / Ph. D. in Biological Chemistry
| Identifer | oai:union.ndltd.org:MIT/oai:dspace.mit.edu:1721.1/115805 |
| Date | January 2018 |
| Creators | Lee, Wankyu |
| Contributors | JoAnne Stubbe., Massachusetts Institute of Technology. Department of Chemistry., Massachusetts Institute of Technology. Department of Chemistry. |
| Publisher | Massachusetts Institute of Technology |
| Source Sets | M.I.T. Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 321 pages, application/pdf |
| Rights | MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission., http://dspace.mit.edu/handle/1721.1/7582 |
Page generated in 0.01 seconds