Spectrin domains are found as multiple tandem repeats in a number of cytoskeleta

Question

Spectrin domains are found as multiple tandem repeats in a number of cytoskeletal proteins such as the spectrins, alpha-actinin and dystrophin. The repeat unit folds into a coiled coil with three antiparallel helices. The structure of the 16th repeat from chicken brain alpha-spectrin (R16) has been determined and is shown as a ribbon diagram. Chevron plots derived from stopped-flow fluorescence experiments on wild-type R16 (WT) and the L108A mutant are also shown below: a. Estimate the difference in stability between the WT and L108A mutant. What assumptions do you make in giving this estimate? b. The unfolding arms of these chevron plots show small but noticeable curvature, indicating a deviation from pure 2-state behavior. Possible explanations include a broad transition state region, or presence of a high-energy on-path intermediate. Is the effect of the mutation L108A present early in the folding pathway, or is this likely a residue that makes many of its interactions late in folding? Explain.

Explanation / Answer

a. The y-axis provides information about the observed rate constant (Kobs) which is significantly lower, under urea, for wild type spectrin in compare to L108A. That is showing the lesser stability of wild type spectrin to L108A.

b. The effect of mutation presents early during the folding pathway because L108A starts to fold in 2.5 M Urea concentration and wild type started to fold at 3.5M urea concentration.

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