Protein-Protein Interactions of the Human Iron Sulfur Cluster Biosynthesis Complex

Iron sulfur (Fe-S) clusters are integral cofactors responsible for a number of cellular processes including electron transfer, catalyzing substrate turnover, sensing small molecules, and regulating gene expression or enzymatic activity. Elaborate multi-component systems have evolved to protect organism from the toxic effects of free iron and sulfide ions while promoting the efficient biosynthesis of these cofactors. Previously, our lab discovered the human cysteine desulfurase complex (NFS1-ISD11, named SD) and the Fe-S assembly scaffold protein ISCU2 form a low activity Fe-S assembly complex (named SDU) that can be activated by the allosteric activator frataxin (FXN) to generate the high activity SDUF Fe-S assembly complex. Importantly, mutations in FXN result in the neurodegenerative disease Friedreich’s Ataxia (FRDA), whereas mutations in ISCU2 lead to a disease characterized by myopathy with exercise intolerance. The goals of this dissertation were to provide structural details for protein-protein interactions in the human SDUF complex, contribute to understanding how FXN binds and activates the assembly complex, and define how a clinical ISCU2 variant was compromised in Fe-S assembly activity. To address these questions, a multidisciplinary approach was initiated that included anaerobic biochemistry and kinetic assays, fluorophore incorporation for anisotropy measurements, and chemical modifications coupled to mass spectrometry experiments. First, protein interfaces were probed by hydroxyl radical footprinting experiments where the SD, SDU, and SDUF complexes were exposed to different does of synchrotron radiation (generating hydroxyl radicals) and the resulting modified proteins were proteolytically digested and analyzed by MALDI mass spectrometry. These experiments revealed that ISCU2 binding to the SD complex results in a decrease in modification kinetics to for regions of ISCU2 near the N-terminus. Consistent with this assignment, kinetic assays revealed that the clinical ISCU2 variant, which has a mutation near the N-terminus, exhibits cysteine desulfurase and Fe-S assembly activities similar to native ISCU2, but compromised binding affinity to the assembly complex. Next, hydroxyl radical footprinting experiments revealed that FXN binding to the SDU complex resulted in the C-terminal α-helix of ISCU2 becoming more protected and suggested specific interactions associated with FXN activation. Next, fluorescence anisotropy experiments under different experimental conditions revealed both determinants for FXN binding and that the FRDA variant has compromised binding affinity to the SDU complex. Finally, this activation model was tested and supported by mutagenesis and binding studies that indicated residues in this C-terminal α-helix of ISCU2 interacts with residues on the β-sheet region of FXN, which are associated with FRDA. Together, these studies reveal details of the protein-protein interactions and function of the human SDUF complex that have implications for human disease

The stoichiometry of the outer kinetochore is modulated by microtubule-proximal regulatory factors

The kinetochore is a large molecular machine that attaches chromosomes to microtubules and facilitates chromosome segregation. The kinetochore includes submodules that associate with the centromeric DNA and submodules that attach to microtubules. Additional copies of several submodules of the kinetochore are added during anaphase, including the microtubule binding module Ndc80. While the factors governing plasticity are not known, they could include regulation based on microtubule–kinetochore interactions. We report that Fin1 localizes to the microtubule-proximal edge of the kinetochore cluster during anaphase based on single-particle averaging of super-resolution images. Fin1 is required for the assembly of normal levels of Dam1 and Ndc80 submodules. Levels of Ndc80 further depend on the Dam1 microtubule binding complex. Our results suggest the stoichiometry of outer kinetochore submodules is strongly influenced by factors at the kinetochore–microtubule interface such as Fin1 and Dam1, and phosphorylation by cyclin-dependent kinase. Outer kinetochore stoichiometry is remarkably plastic and responsive to microtubule-proximal regulation

Primary Sequence Confirmation of a Protein Therapeutic Using Top Down MS/MS and MS³

Mass spectrometry has gained widespread acceptance for the characterization of protein therapeutics as a part of the regulatory approval process. Improvements in mass spectrometer sensitivity, resolution, and mass accuracy have enabled more detailed and confident analysis of larger biomolecules for confirming amino acid sequences, assessing sequence variants, and characterizing post translational modifications. This work demonstrates the suitability of a combined approach using intact MS and multistage top down MS/MS analyses for the characterization of a protein therapeutic drug. The protein therapeutic granulocyte-colony stimulating factor was analyzed using a Thermo Fusion Tribrid mass spectrometer using a multistage top down MS approach. Intact mass analysis identified the presence of two disulfide bonds based on exact mass shifts while a combined collision induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron transfer dissociation (ETD) MS/MS approach obtained 80% protein sequence coverage. Isolating MS/MS fragments for MS³ analysis using HCD or CID increased the sequence coverage to 89%. 95% sequence coverage was obtained by reducing human granulocyte-colony stimulating factor (G-CSF) prior to MS/MS and MS³ analysis to specifically target the residues between the disulfide bonds. The use of this combined intact MS and multistage top down MS approach allows for rapid and accurate determination of the primary sequence of a protein therapeutic drug product

Analysis of Fluorescent Proteins with a Nanoparticle Probe

This Letter presents the first application of high-energy, single nanoparticle probes (e.g., 520 keV Au₄₀₀ 2 nm NP) in the characterization of surfaces containing fluorescent proteins (e.g., GFP variants) by their coemitted photon, electron and secondary ion signals. NP-induced protein luminescence increases with the NP incident energy, is originated by the NP impact, and is transferred to the protein fluorophor via electronic energy transfer. Multielectron emission is observed per single NP impacts, and their distributions are specific to the target morphology and composition. Fragment ions of protein subunits consisting of 2–7 amino acid peptides are observed under individual NP impacts that can be correlated to the random protein orientation relative to the impact site (e.g., outer layer or “skin” of the protein)

Analysis of Fluorescent Proteins with a Nanoparticle Probe

This Letter presents the first application of high-energy, single nanoparticle probes (e.g., 520 keV Au400 2 nm NP) in the characterization of surfaces containing fluorescent proteins (e.g., GFP variants) by their coemitted photon, electron and secondary ion signals. NP-induced protein luminescence increases with the NP incident energy, is originated by the NP impact, and is transferred to the protein fluorophor via electronic energy transfer. Multielectron emission is observed per single NP impacts, and their distributions are specific to the target morphology and composition. Fragment ions of protein subunits consisting of 2-7 amino acid peptides are observed under individual NP impacts that can be correlated to the random protein orientation relative to the impact site (e.g., outer layer or "skin" of the protein)