?url_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Adc&rft.relation=http%3A%2F%2Fd-scholarship-dev.library.pitt.edu%2F12184%2F&rft.title=Probing+the+Structure+and+Dynamics+of+a+Protein-DNA+Complex+Using+Electron+Spin+Resonance&rft.creator=Sarver%2C+Jessica&rft.description=In+this+thesis%2C+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%2C+due+to+its+high+binding+specificity%2C+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%2C+the+length+and+flexibility+of+the+spin+label+convolute+these+distance+measurements%2C+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+%CE%B1-helix+and+a+%CE%B2-strand.++In+addition%2C+we+used+the+simulations+to+gain+information+on+the+backbone+motion+at+the+spin-labeled+site+by+measuring+the+C%CE%B1-C%CE%B1+distributions.%0D%0ADynamics+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%2C+miscognate+(one+base+pair+mismatch)+and+nonspecific+(%3E2+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.++%0D%0ALastly%2C+this+thesis+discusses+the+comparison+of+two+distance+measurement+techniques+for+a+metal-nitroxide+system.++We+performed+distance+measurements+between+a+bound+Cu2%2B+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%2C+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.%0D%0A&rft.date=2012-10-02&rft.type=University+of+Pittsburgh+ETD&rft.type=PeerReviewed&rft.format=application%2Fpdf&rft.language=en&rft.identifier=http%3A%2F%2Fd-scholarship-dev.library.pitt.edu%2F12184%2F1%2FJessica_Sarver_-_Thesis%25282%2529_-_Chemistry.pdf&rft.identifier=++Sarver%2C+Jessica++(2012)+Probing+the+Structure+and+Dynamics+of+a+Protein-DNA+Complex+Using+Electron+Spin+Resonance.++Doctoral+Dissertation%2C+University+of+Pittsburgh.++++(Unpublished)++