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INVESTIGATION OF PROTEIN STRUCTURE AND DYNAMICS BY NMR SPECTROSCOPY

Unnikrishnan, Aparna
http://orcid.org/0000-0002-6668-8659
http://rave.ohiolink.edu/etdc/view?acc_num=osu1595418229203869

Abstract Details

2020, Doctor of Philosophy, Ohio State University, Biophysics.
The revelation of multiple metastable conformational states in proteins, has led to a paradigm shift in the understanding of protein structure-function relationships. Nuclear magnetic resonance (NMR) spectroscopy-based methodologies allow us to gain significant insights into how the intrinsic flexibility of protein molecules regulate their function, thus enhancing our understanding of regulatory pathways that can be explored to attain greater mechanistic control. I have applied NMR spectroscopy to characterize two different types of biomolecules that are directly, or indirectly, capable of gene modification. The Cre recombinase enzyme investigated in Chapters 2 and 3 is a powerful mammalian genetic engineering tool capable of precise DNA cleavage and exchange, and the Brd4 ET domain that is discussed in Chapter 4 is an epigenetic reader oncoprotein involved in the recruitment of other gene/expression modifying proteins to chromatin, and is implicated in many disease states. The mechanism of DNA recombination by Cre recombinase has been illuminated so far by crystallographic snapshots of tetrameric synaptic reaction intermediates. Cre becomes active within the tetrameric scaffold but remains inactive as a monomer. The lack of information on the structural and dynamic features of Cre recombinase in its free form and in other pre-synaptic intermediate states has left a gap in our understanding of the mechanisms that regulate its function. The observation of conformational plasticity in the catalytic domain of Cre led us to hypothesize a regulatory role of intrinsic protein flexibility. Remarkably, while characterizing the dynamics in the domain using solution NMR techniques, we discovered a cis to trans switch in the C-terminal region of Cre, that may act to auto-inhibit the protein prior to DNA binding, and subsequently enables the protein to undergo assembly of tetrameric intasomes on the bound DNA. Solution NMR data revealed that a C-terminal segment of the protein, which adopts an extended helix in crystal structures of synaptic complexes refolds to form a new anti-parallel beta-sheet to the Cre interdomain linker, thereby blocking accessibility to the protein active site. The interaction between the chromatin recruiter domain Brd4 ET and the histone methyl transferase protein NSD3 had been previously speculated to follow a conserved mechanism, involving binding-induced folding to form an inter-molecular anti-parallel beta-sheet. A short four-residue peptide from NSD3 was meanwhile shown to be essential to the interaction with Brd4 ET. I performed biophysical experiments to characterize this interaction and to identify the minimal region of NSD3 that constitutes the entire Brd4 ET binding surface. Using NMR, isothermal titration calorimetry, mass-spectrometry based protein footprinting, and affinity pull down assays, we found that the reported short peptide region of NSD3 is insufficient to constitute the entire NSD3-ET binding region. This indicates the involvement of other regions of NSD3 in interaction with Brd4 ET domain, and paves the way for future efforts towards development of therapeutics targeting this protein-protein interaction.
Mark Foster (Advisor)
Charles Bell (Committee Member)
Thomas Magliery (Committee Member)
197 p.
Biochemistry; Biophysics
Solution NMR, Protein dynamics, Paramagnetic Relaxation Enhancement, cis to trans conformational switch

Recommended Citations

Citations

  • Unnikrishnan, A. (2020). INVESTIGATION OF PROTEIN STRUCTURE AND DYNAMICS BY NMR SPECTROSCOPY [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1595418229203869

    APA Style (7th edition)

  • Unnikrishnan, Aparna. INVESTIGATION OF PROTEIN STRUCTURE AND DYNAMICS BY NMR SPECTROSCOPY. 2020. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1595418229203869.

    MLA Style (8th edition)

  • Unnikrishnan, Aparna. "INVESTIGATION OF PROTEIN STRUCTURE AND DYNAMICS BY NMR SPECTROSCOPY." Doctoral dissertation, Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1595418229203869

    Chicago Manual of Style (17th edition)

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© 2020, some rights reserved.Creative Commons License INVESTIGATION OF PROTEIN STRUCTURE AND DYNAMICS BY NMR SPECTROSCOPY by Aparna Unnikrishnan is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by The Ohio State University and OhioLINK.