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University of Toronto St. George
Dave Tulumello

LECTURE 03Anfinsens experiment Results with ribonuclease led to the main conclusion thato Sequence specifies conformationo AKA primary sequence determines secondary and tertiary structure Renaturation of ribonucleaseDenatured reduced ribonuclease undergoes dialysis to remove urea and betamercaptoethanol then undergoes air oxidation of the sulfhydryl groups in reduced ribonuclease RESULTS in native ribonuclease o During dialysis only small molecules can traverse the semipermeable membrane Refolding of ribonucleaseAfter dialysis the enzyme slowly regains activity The sulfhydryl groups become reoxidized by air to form the refolded catalyticallyactive form Reduction and denaturation of ribonucleaseNative ribonuclease becomes denatured reduced ribonuclease Denaturing agent ureaNH2CONH2o This is able to form hydrogen bonds with protein backbone Protein folding pathway using cardboard box modelUnfolded undergoes several intermediates and transition states to become native folded foxIn the unfolded state the flaps of the protein are free to move independentlyEntropy is lost during folding A large energy barrier must be surmounted to convert from almostfolded to fullyfolded o This is to ensure that proteins do not easily denature Folding transitionbox can go backwards in the pathway ie become unfolded as it originally wasNative state of proteinso Native state with all interactions is only marginally stable relative to the unfoleddenatured state BUT possesses considerable kinetic stability because it can unfold only by passing through a highenergy transition state in which most native interactions are disrupted simultaneously Native RNase formed from scrambled RNase in the presence of betamercaptoethanol Scrambled to native Anfinsens experiment When RNase was refoled in 8M urea by oxidation and then dialyzed to remove urea only 1 enzymatic activity was foundo bc wrong disulfide pairing occurringo there are 105 ways to pair 8 cysteines into 4 disulfide pairsAnalysis of Proteins PURIFICATION BY CHROMATOGRAPHY
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