A Minimum Folding Unit in the Ankyrin Repeat Protein p16^INK4

ISSN： 0022-2836

Source： Journal of Molecular Biology, Vol.299, Iss.4, 2000-06, pp. : 1121-1132

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Abstract

The ankyrin repeat is an abundant, 33 residue sequence motif that forms a consecutive &bgr;-hairpin-helix-loop-helix (&bgr;₂₂) fold. Most ankyrin repeat proteins consist of four or more complete repeats, which provide stabilizing interactions between adjacent modules. The cyclin-dependent kinase inhibitor and tumor suppressor p16^INK4 (p16) is one of the smallest ankyrin repeat proteins with a known structure. It consists of four complete repeats plus short N and C-terminal flanking regions that are unstructured in solution. On the basis of preliminary proteolysis studies and predictions using a computer algorithm for identifying autonomous folding units, we have identified a fragment consisting of the third and fourth ankyrin repeats of p16, called p16C, that can fold independently, without the rest of the protein. Far-UV circular dichroism studies showed that p16C has a significant level of -helical secondary structure, and two proline substitutions that disrupt the -helical secondary structure in wild-type p16 disrupt the secondary structure in p16C. The thermal denaturation of p16C is cooperative and reversible, with a midpoint of transition at 30.5(±1)°C. From urea-induced denaturation studies, the free energy of unfolding for p16C was estimated to be 1.7(±0.3) kcal/mol at 20 °C. ¹H-¹⁵N 2D NMR studies suggest that the ankyrin repeats in p16C are likely to fold into a structure similar to that of full-length p16. In order to define the minimum autonomous folding unit in p16, we have further dissected p16C into two complementary peptides, each containing a single ankyrin repeat. These peptides are unstructured in solution. Thus, p16C is the smallest ankyrin repeat module that is known to fold independently and, in general, we believe that the two-ankyrin repeat fold could be the minimum structural unit for all ankyrin repeat proteins. We further discuss the significance of p16C in protein folding and engineering.