J Michael Sauder - Amide protection in an early folding intermediate of cytochrome c

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      Publication Details (including relevant citation   information):

      Folding & Design (1998) 3: 293-301.

      Sauder JM, Roder H


      BACKGROUND: For many proteins, compact states appear long before   the  rate-limiting step in the formation of the native   structure. A key issue  is whether the initial collapse of   the chain is driven by random or  more specific hydrophobic   interactions. RESULTS: Hydrogen-exchange  labeling coupled   with NMR was used to monitor the formation of stable    hydrogen-bonded and solvent-excluded structure in horse   cytochrome c  (cyt c). Protection was measured using a   hydrogen exchange/folding  competition protocol at variable   pH and short competition time (2 ms).  Protection factors of   threefold to eightfold were observed in all three  alpha   helices of cyt c, whereas other regions showed no   significant  protection. This suggests that the compact   states that are present  contain segments of marginally   stable hydrogen-bonded structure. When  the intermediate(s)   are destabilized, only amide protons from Cys14,  Ala15 and   His18 show significant protection, indicating a region of    persistent residual structure near the covalently bound heme   group in  the unfolded protein. Fluorescence-detected   stopped-flow studies showed  that the maximum protection   factor in the early intermediate is  consistent with its   unfolding equilibrium constant. CONCLUSIONS:  Together with   previous fluorescence and CD results, the observed pattern    of amide protection is consistent with the early formation of   an  alpha-helical core domain in an ensemble of compact   states, indicating  that efficient folding is facilitated by   stepwise acquisition of native  structural elements. These   specific early interactions are established  on the   sub-millisecond time scale, prior to the rate-limiting step   for  folding.

      Address (URL): http://www.ncbi.nlm.nih.gov/pubmed/9710575