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Folding-TIM Barrel Protein Folding Practical September 2011 Folding up the TIM barrel Preliminary Examine the parallel beta barrel that you constructed, noting the stagger of the strands that was needed to connect the ends of the 8-stranded parallel beta sheet into the 8-stranded beta barrel. Notice that the stagger dictates which side of the sheet is on the inside and which is on the outside. This will be key information in folding the complete TIM linear peptide into the TIM barrel. Assembling the full linear peptide 1. Make sure the white beta strands are extended correctly, and the 8 yellow helices (with the green loops at each end) are correctly folded into an alpha helix (right handed with H-bonds to the 4th ahead in the chain). 2. starting with a beta strand connect an alpha helix and green loop to make the blue-red connecting peptide bond. Making sure that you connect the carbonyl (red) end of the beta strand to the amino (blue) end of the loop-helix-loop. Secure the just connected peptide bond bond with a twist-tie as shown. 3. complete step 2 for all beta strand/loop-helix-loop pairs, working in parallel with your partners 4. As pairs are completed attach the carboxy end of the strand- loop-helix-loop to the amino end of the next strand-loop-helix-loop module and secure the new peptide bond with a twist-tie as before. Repeat until the full linear TIM polypeptide chain is assembled. Make sure all strands and helices are still in the correct conformations. Folding the TIM superhelix The folding of the final TIM barrel makes a right handed super-helical structure with the alternating strand- loop-helix motif. By recalling the inside and outside of the previously folded parallel beta barrel, do the following: 1. start at the amino terminus of the linear polypeptide and tuck the first helix under the beginning strand so that the next beta strand can hydrogen-bond to the first in a Protein Folding Practical September 2011 parallel fashion. At this point hydrogen bond the first two strands and check to see if forms a right handed helix. Also check to see if the bonded strands are staggered in the correct direction (so that the helix will be on the outside of the barrel). If the stagger is too large, the attached beta strand must be rolled over so that the alpha carbon stubs facing the outside (down) are now facing the inside (up). This will adjust the stagger to the correct spacing when the hydrogen bonds are re-made. 2. Continue as in step 2 by folding under the next helix and hydrogen bonding the next beta strand on top, continuing the same stagger. 3. When all 8 beta strands are connected into a sheet, the TIM superhelix will be laying flat on the table with the beta sheet on top of the 8 alpha helices. The pattern should look the same as the original 8 stranded beta sheet that was folded into a regular beta barrel. Folding up the TIM barrel Carefully lift the two ends of the 8 stranded beta sheet with the attached alpha helices, leaving the carboxy terminal helix hanging. Cradle the structure with two hands and as you attach the two end strands together by hydrogen bonding them with the same stagger as the others into the closed barrel. Make sure all of the beta barrel hydrogen bonds are still intact. Stand the barrel up and place the carboxy terminal helix against the beta barrel in the same orientation as the other helices. .
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