Unraveling Macro-Molecular Machinery by Mass Spectrometry: from Single Proteins to Non-Covalent Protein Complexes
AdvisorWysocki, Vicki H.
Committee ChairWysocki, Vicki H.
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PublisherThe University of Arizona.
RightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
AbstractPresented in this dissertation are studies of protein dynamics and protein/protein interactions using solution phase hydrogen/deuterium exchange in combination with mass spectrometry (HXMS). In addition, gas phase fragmentation behaviors of deuterated peptides are investigated, with the purpose of increasing resolution of the HXMS. In the area of single protein dynamics, two protein systems are studied. Studies on the cytochrome c2 from Rhodobacter capsulatus indicate its domain stability to be similar to that of the horse heart cytochrome c. Further comparison of the exchange kinetics of the cytochrome c2 in its reduced and oxidized state reveals that the so-called hinge region is destabilized upon oxidation. We also applied a similar approach to investigate the conformational changes of photoactive yellow protein when it is transiently converted from the resting state to the signaling state. The central β-sheet of the protein is shown to be destabilized upon photoisomerization of the double bond in the chromophore. Another equally important question when it comes to understanding how proteins work is the interactions between proteins. To this end, two protein complexes are subjected to studies by solution phase hydrogen deuterium exchange and mass spectrometry. In the case of LexA/RecA interaction, both proteins show decreases in their extents of exchange upon complex formation. The potential binding site in LexA was further mapped to the same region that the protein uses to cleave itself upon interacting with RecA. In the sHSP/MDH system, hydrogen/deuterium exchange experiments revealed regions within sHSP-bound MDH that were significantly protected against exchange under heat denaturing condition, indicative of a partially unfolded state. Hydrogen/deuterium exchange therefore provides a way of probing low resolution protein structure within protein complexes that have a high level of heterogeneity. Finally, the feasibility of increasing resolution of HXMS by gas phase peptide fragmentation is investigated by using a peptide with three prolines near the C-terminus. Our data show that deuterium migration indeed occurs during the collision activated dissociation process. Caution is required when interpreting the MS/MS spectra as a way of pinpointing the exact deuterium distribution within peptides.
Degree GrantorUniversity of Arizona
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Protein stickiness, rather than number of functional protein-protein interactions, predicts expression noise and plasticity in yeastBrettner, Leandra M.; Masel, Joanna; Present address: Ecology & Evolutionary Biology, University of Arizona, 1041 E Lowell St, Tucson, AZ, 85721, USA; Present address: Bioengineering, University of Washington, 3720 15th Ave NE, Seattle, WA, 98195, USA (BioMed Central, 2012)BACKGROUND:A hub protein is one that interacts with many functional partners. The annotation of hub proteins, or more generally the protein-protein interaction "degree" of each gene, requires quality genome-wide data. Data obtained using yeast two-hybrid methods contain many false positive interactions between proteins that rarely encounter each other in living cells, and such data have fallen out of favor.RESULTS:We find that protein "stickiness", measured as network degree in ostensibly low quality yeast two-hybrid data, is a more predictive genomic metric than the number of functional protein-protein interactions, as assessed by supposedly higher quality high throughput affinity capture mass spectrometry data. In the yeast Saccharomyces cerevisiae, a protein's high stickiness, but not its high number of functional interactions, predicts low stochastic noise in gene expression, low plasticity of gene expression across different environments, and high probability of forming a homo-oligomer. Our results are robust to a multiple regression analysis correcting for other known predictors including protein abundance, presence of a TATA box and whether a gene is essential. Once the higher stickiness of homo-oligomers is controlled for, we find that homo-oligomers have noisier and more plastic gene expression than other proteins, consistent with a role for homo-oligomerization in mediating robustness.CONCLUSIONS:Our work validates use of the number of yeast two-hybrid interactions as a metric for protein stickiness. Sticky proteins exhibit low stochastic noise in gene expression, and low plasticity in expression across different environments.
Methods for the Detection of Protein-Nucleic Acid and Protein-Protein InteractionsGhosh, Indraneel; Stains, Cliff; Ghosh, Indraneel; Hruby, Victor J.; McGrath, Dominic V.; Montfort, William R. (The University of Arizona., 2008)We describe the first general approach for the DNA templated reassembly of proteins, which we term SEquence-Enabled Reassembly or SEER. SEER makes use of dissected signaling domains which are each attached to separate, sequence specific DNA-binding proteins. Described herein is an embodiment of SEER in which DNA catalyzes the reassembly of the green fluorescent protein which leads to a direct fluorescence readout of the corresponding DNA sequence. This strategy has also been extended to the first direct method for the site specific detection of DNA methylation. This mCpG-SEER system is capable of discriminating between methylated versus nonmethylated DNA with a 40-fold increase in fluorescence signal.In a separate undertaking we tested the efficiency of disulfide bond formation within the context of the ribosome display in vitro selection methodology. We established conditions for the enrichment of a cyclic peptide, which is specific for Neutravidin, by 2 x 10^6-fold. Using the knowledge gained from the above experiments, we combined the rapid protein expression and folding benefits of cell-free translation systems with a sensitive split-luciferase reassembly assay to yield the most rapid method to date for the detection of protein-nucleic acid and protein-protein interactions. Furthermore, we have shown that these split-luciferase cell-free reassembly systems can be compartmentalized, allowing for future molecular evolution studies.Lastly, we have applied this rapid cell-free split-luciferase assay system to the direct detection of clinically relevant proteins. We have engineered a system for the rapid characterization of HIV-1 clades utilizing single-chain antibody specificities. We also demonstrate that this platform can be used to determine the relative amounts of HER2 expression in human breast cancer cells, using a homogeneous assay format in which cells and reagents are mixed and luminescence is monitored directly.We envision that the assay platforms described herein will find applications in the rapid detection of nucleic acid sequences, protein identities, and relative protein abundances in the laboratory and clinic.
PROTEIN-PROTEIN INTERACTIONS OF HUMAN PARVOVIRUS B19 NS1 AND IDENTIFICATION OF THE NS1 TRANSCRIPTIONAL TRANSACTIVATION DOMAINHorton, Nancy; Morano, Alexandra Lynn (The University of Arizona., 2018)Infection with human parvovirus B19V (B19V) has been associated with a myriad of illnesses, including erythema infectiosum (Fifth disease), hydrops fetalis, arthropathy, hepatitis, cardiomyopathy and also possibly the triggering of any number of different autoimmune diseases. B19V NS1 is a multidomain protein that plays a critical role in viral replication, with predicted nuclease, helicase, and gene promoter transactivation activities. Herein we utilize a yeast two-hybrid assay to identify human proteins that bind to B19V NS1. We discovered 26 new human proteins involved in diverse pathways including transcription activation, transcription repression, viral response, immune regulation, stress granule response, transmembrane transport, heme synthesis, apoptosis, and cell cycle among others. All but one of these interactions were lost with truncations of NS1 to only its N-terminal nuclease domain, or removal of the C-terminal domain. In addition, we determine that the C-terminal domain of B19V NS1 harbors gene promoter transcriptional transactivation activity, and that this activity is sequestered or otherwise inactivated in full length NS1 when expressed in yeast. Loss of the ATPase activity via mutation in NS1 did not change sequestration, but did result new protein-protein interactions not seen with wild type NS1.