eprintid: 12184 rev_number: 20 userid: 955 dir: disk0/00/01/21/84 datestamp: 2012-10-02 19:08:44 lastmod: 2016-11-15 13:58:17 status_changed: 2012-10-02 19:08:44 type: thesis_degree metadata_visibility: show contact_email: sarver383@gmail.com item_issues_count: 0 eprint_status: archive creators_name: Sarver, Jessica creators_email: jls144@pitt.edu creators_id: JLS144 title: Probing the Structure and Dynamics of a Protein-DNA Complex Using Electron Spin Resonance ispublished: unpub divisions: sch_as_chemistry full_text_status: public keywords: Electron Spin Resonance, Molecular Dynamics Simulation, Protein-DNA Complex, Spin Label abstract: In this thesis, we employ site-directed spin labeling and electron spin resonance to probe the changes in structure and dynamics for different EcoRI-DNA complexes. EcoRI is a restriction endonuclease that, due to its high binding specificity, is a model system for investigating protein-DNA interactions. Distance constraints were previously obtained for spin-labeled mutants of EcoRI in complex with DNA. However, the length and flexibility of the spin label convolute these distance measurements, making it difficult to extract biologically relevant information. We performed molecular dynamics (MD) simulations on the spin-labeled EcoRI-DNA complex to understand the packing and dynamics of the spin label. We show that correlated distance constraints can be leveraged with MD simulations to learn about the preferred conformers of the spin label on sites in an α-helix and a β-strand. In addition, we used the simulations to gain information on the backbone motion at the spin-labeled site by measuring the Cα-Cα distributions. Dynamics and order may play a role in the DNA binding specificity of EcoRI. We performed continuous wave (CW) experiments to measure the dynamics and order of EcoRI bound to its specific, miscognate (one base pair mismatch) and nonspecific (>2 base pair mismatch) DNA sites. We analyzed CW spectra for three EcoRI complexes spin-labeled at various sites in a region of the protein that is believed to play a role in the binding specificity of EcoRI. Simulation of the spectra provided details on the change in dynamics and order in the different complexes. We found the protein-DNA interface to be most sensitive to the changes between the complexes. Spin-labeled sites further from the DNA were found to show little variation in dynamics between the complexes. Lastly, this thesis discusses the comparison of two distance measurement techniques for a metal-nitroxide system. We performed distance measurements between a bound Cu2+ and a spin label on two polyalanine peptides of differing length using DEER and relaxation-based saturation recovery experiments. By comparing the results of both measurements, we show that the relaxation-based technique is biased to shorter average distances. This work highlights the advantages of using DEER to perform metal-nitroxide distance measurements. date: 2012-10-02 date_type: published pages: 169 institution: University of Pittsburgh refereed: TRUE etdcommittee_type: committee_chair etdcommittee_type: committee_member etdcommittee_type: committee_member etdcommittee_type: committee_member etdcommittee_name: Saxena, Sunil etdcommittee_name: Waldeck, David etdcommittee_name: Weber, Stephen etdcommittee_name: Jen-Jacobson, Linda etdcommittee_email: sksaxena@pitt.edu etdcommittee_email: dave@pitt.edu etdcommittee_email: sweber@pitt.edu etdcommittee_email: ljen@pitt.edu etdcommittee_id: SKSAXENA etdcommittee_id: DAVE etdcommittee_id: SWEBER etdcommittee_id: LJEN etd_defense_date: 2012-05-02 etd_approval_date: 2012-10-02 etd_submission_date: 2012-05-21 etd_release_date: 2012-10-02 etd_access_restriction: immediate etd_patent_pending: FALSE assigned_doi: doi:10.5195/pitt.etd.2012.12184 thesis_type: dissertation degree: PhD citation: Sarver, Jessica (2012) Probing the Structure and Dynamics of a Protein-DNA Complex Using Electron Spin Resonance. Doctoral Dissertation, University of Pittsburgh. (Unpublished) document_url: http://d-scholarship-dev.library.pitt.edu/12184/1/Jessica_Sarver_-_Thesis%282%29_-_Chemistry.pdf