Lecture 10 - Lecture 10 Friday Announcements 1 Quiz 2A 24.8...

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Lecture 10 Friday, September 15, 2006 Announcements 1. Quiz 2A 24.8 Quiz 2B 24.3 Quiz 2C 25.2 2. We do not assign letter grades at this time. To give you a very rough idea of where you stand: divide your raw score by the mean to find your normalized score ("NS"); use this very rough guide: mid-range for grade of A, NS = 1.2; mid- range for grade of B+, NS = 1.0; mid-range for grade of C, NS = 0.80. This is a rough guide . 3. Three Wednesdays when there will NOT be a quiz: 10/11 (day of return from Fall Break) 10/18 (day before the midterm exam) 11/22 (day when Thanksgiving recess begins) 4. Meet today with any interested students to discuss “Should I apply to medical school this year? Should I apply at all!? Help!” Comstock B106, 1:25 – 2:15PM. 5. Have a Rasmol problem? Send query to the Rasmol TA. If you send to the VOH, you will introduce a delay in our response. Wednesday's lecture: Thermodynamic treatment of protein folding/unfolding and representation of stability using a graph of G o vs “structural coordinates”. Today's lecture: Let’s look at two proteins, ribonuclease and insulin. Looking at page 75, you see that both of these proteins have disulfide bonds . To denature these proteins, add 8M urea (to "destroy" the normal water H- bond structure that drives the hydrophobic interaction), and also add something that will reduce the disulfide bonds . We add mercaptoethanol. In both cases, after this treatment we get unfolded, inactive protein. To attempt renaturation, dialyze to remove the urea. Then allow mild air oxidation, so that the disulfide bonds can reform. In the case of ribonuclease, after dialysis and air oxidation, we have full biological activity, but it takes 10 hours to form! In the cell, it takes about 2 minutes for ribonuclease to fold and form its disulfides.
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In the case of insulin, after dialysis, we are not sure what the structure is. After oxidation, there is little to no biological activity! In both cases, folding occurred to give the most stable final structure-- the lowest G o of (polypeptide + water) under the folding conditions. Then why did not the native insulin structure re-form? Original synthesis of insulin on the ribosome creates one chain called pre-pro- insulin, 107 AA long. After synthesis inside the cell, a small 23 AA piece of the polypeptide is cleaved off from one end. This piece is the "signal" that targets insulin to cross a membrane, necessary because insulin must be packaged inside membrane vesicle for secretion. This cleavage yields pro-insulin, which is 84 AA long. Proinsulin folds spontaneously to minimize the standard free energy. It now has the “correct structure,” but it is not done yet... Finally, a piece of the pro-insulin, 33AA long, in the middle of the molecule, is cleaved out, and the result is native, biologically active insulin. But some information has been lost - some of the folding information that was critical to proper folding! The information that was cleaved out is needed in order for proper folding that will result in 100% activity. The lesson here is that, in general,
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  • Summer '07
  • FEIGENSON,GW
  • Proteins

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