?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%2F39679%2F&rft.title=Site-Directed+Cu2%2B+Labeling+Methodologies+for+Obtaining+Distance+Constraints+in+Proteins+and+DNA&rft.creator=Ghosh%2C+Shreya&rft.description=Electron+spin+resonance+(ESR)+spectroscopy+along+with+site-specific+introduction+of+spin+labels+into+macromolecules+is+a+powerful+tool+to+obtain+a+multifaceted+view+of+macromolecule+structure%2C+flexibility%2C+and+dynamics.+In+this+dissertation%2C+we+develop+Cu2%2B-based+ESR+reporters+for+proteins+and+DNA+that+are+small%2C+rigid+and+can+provide+precise+information+on+the+backbone+fluctuations+without+requiring+additional+modeling.+First%2C+we+demonstrate+a+nucleotide+and+structure-independent+Cu2%2B-based+label+for+DNA%2C+that+can+be+incorporated+anywhere+in+the+DNA+duplex.+We+perform+pulsed+ESR+based+distance+measurements+on+several+duplexes+with+varying+base+pair+separation+between+the+labels.+Using+the+distance+measurements+along+with+modeling%2C+we+illustrate+that+this+methodology+is+capable+of+directly+reporting+on+DNA+backbone+conformations+in+solution.+Additionally%2C+we+perform+molecular+dynamics+simulations+using+high-quality+force+field+parameters+developed+for+the+Cu2%2B-label+in+DNA.+Such+analysis+provides+detailed+atomic+insights+into+the+conformational+fluctuations+of+the+label+and+a+more+nuanced+picture+of+the+ESR+distance+measurements.+%0D%0AFor+proteins%2C+we+further+develop+a+labeling+strategy+where+Cu2%2B+site-specifically+binds+to+two+strategically+placed+histidine+residues+in+a+protein.+Systematic+analysis+confirms+that+the+Cu2%2B-complex+binds+specifically+to+the+engineered+binding+sites.+Distance+measurements+using+this+label+show+an+improved+resolution+with+a+two-fold+increase+in+the+signal-to-noise+ratio.+The+small+size+and+the+rigidity+of+the+Cu2%2B-label+shows+promise+over+traditional+labels+and+will+allow+for+readily+elucidating+protein+backbone+flexibility%2C+distinguishing+between+different+protein+conformations+in+solution+and+determining+relative+orientations+of+different+protein+subunits.+In+addition%2C+we+also+apply+Cu2%2B-based+ESR+measurements+to+understand+the+influence+of+metal+binding+on+the+homodimeric+antimicrobial+protein%2C+Calgranulin+C.+We+demonstrate+that+despite+being+homodimeric%2C+the+two+native+Cu2%2B+binding+sites+in+Calgranulin+C+have+different+coordination+environments+in+solution%2C+where+only+one+of+the+Cu2%2B+shows+backbone+coordination.+Finally%2C+ESR+distance+measurements+on+the+Cu2%2B-bound+protein+provide+multiple+distances+indicating+metal-induced+protein+oligomerization+in+solution.+Overall%2C+this+dissertation+highlights+the+advantages+of+Cu2%2B-based+labeling+strategies+in+proteins+and+DNA+that+can+find+application+for+understanding+conformational+changes+associated+with+protein-DNA+interactions+in+important+physiological+functions.&rft.date=2021-01-20&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%2F39679%2F21%2FGhosh%2520Final%2520ETD.pdf&rft.identifier=++Ghosh%2C+Shreya++(2021)+Site-Directed+Cu2%2B+Labeling+Methodologies+for+Obtaining+Distance+Constraints+in+Proteins+and+DNA.++Doctoral+Dissertation%2C+University+of+Pittsburgh.++++(Unpublished)++