?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%2F34466%2F&rft.title=Site-Directed+Cu2%2B+Labeling+of+DNA+and+Proteins&rft.creator=Lawless%2C+Matthew&rft.description=Electron+spin+resonance+(ESR)+in+combination+with+site-directed+spin+labeling+can+be+used+to+determine+structure%2C+flexibility+and+conformational+dynamics+of+proteins+and+DNA%2FRNA.+This+thesis+focuses+on+using+Cu2%2B+as+a+spin+label.+First%2C+we+introduce+a+new+method+to+incorporate+Cu2%2B+within+DNA+for+the+purpose+of+creating+an+accurate+reporter+of+DNA+structure.+This+method+positions+the+paramagnetic+label+within+the+interior+of+the+duplex+as+opposed+to+all+current+other+labeling+strategies.+This+methodology+is+also+nucleotide+and+structure+independent.+Using+this+approach%2C+the+measured+interspin+distance+is+within+1+%C3%85+of+the+distance+predicted+by+modeling+and+molecular+dynamics+simulations.+This+method+is+capable+of+reporting+backbone-backbone+distances+without+modeling.%0D%0ACu2%2B-based+strategies+are+also+used+to+measure+protein+structure.+We+measure%2C+by+direct+spectroscopic+measurements%2C+the+optimum+conditions+to+load+Cu2%2B+to+engineered+binding+sites+in+both+%CE%B1-helices+and+%CE%B2-sheets.+The+optimizing+loading+conditions+lead+to+a+two-fold+increase+in+signal+for+the+measurement+of+Cu2%2B-Cu2%2B+distances.+The+procedure+is+then+used+investigate+the+human+glutathione+S-transferase+A1-1+protein.+This+enzyme+promiscuously+binds+to+and+activates+various+forms+of+glutathione+to+reduce+cellular+radical+species.+We+present+a+combination+of+nitroxide-+and+Cu2%2B-based+spin+labeling+to+probe+the+conformation+of+the+functionally+important+terminal+helix.+We+find+that+the+terminal+helix+exists+in+two+distinct+conformations%2C+one+of+which+is+largely+dynamic.+Such+flexibility+might+be+important+for+the+high+degree+of+substrate+promiscuity.+ESR+measurements+are+then+used+as+distance+constraints+to+generate+a+model+of+the+whole+length+protein.+%0D%0AWe+next+developed+strategies+to+perform+structural+measurements+of+proteins+in+their+native+in-cell+environment.+Measurements+in-cell+are+challenging+as+the+ESR+signal+quickly+degrades+within+the+cellular+environment.+To+extend+the+applicability+of+ESR+in-cell%2C+first%2C+we+prolong+the+in-cell+half-life+of+the+typical+ESR+spin+label+with+an+oxidizing+agent.+Second%2C+we+use+a+combination+of+spectroscopic+measurements+to+analyze+the+contribution+of+label+degradation+toward+overall+signal+loss.+Third%2C+we+collect+time-based+data+to+quantify+the+time+required+for+molecular+diffusion+of+a+small+globular+protein+within+the+cellular+milieu.&rft.date=2018-06-28&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%2F34466%2F1%2FThesis%2520Total_final3.pdf&rft.identifier=++Lawless%2C+Matthew++(2018)+Site-Directed+Cu2%2B+Labeling+of+DNA+and+Proteins.++Doctoral+Dissertation%2C+University+of+Pittsburgh.++++(Unpublished)++