Long-range effects and functional consequences of stabilizing mutations in the ankyrin repeat domain of I?B?.

ABSTRACT: Protein domains containing three or more ankyrin repeats (ARs) are ubiquitous in all phyla. Sequence alignments previously identified certain conserved positions, which have been shown to stabilize AR domains and promote their folding. Consensus mutations [Y254L/T257A (YLTA) and C186P/A220P (CPAP)] stabilize the naturally occuring AR domain of human I?B? to denaturation; however, only the YLTA mutations stabilize the protein to proteasomal degradation. We present results from NMR experiments designed to probe the roles of these consensus mutations in I?B?. According to residual dipolar coupling analysis, the gross structures of the AR domains of both mutants appear to be similar to the wild type (WT). Comparison of chemical shifts of mutant and WT proteins reveals that the YLTA and CPAP consensus mutations cause unexpected long-range effects throughout the AR domains. Backbone dynamics experiments reveal that the YLTA mutations in the sixth AR order the C-terminal PEST sequence on the picosecond-to-nanosecond timescale, compared to either the WT or the CPAP mutant I?B?s. This property is likely the mechanism by which the half-life of YLTA I?B? is extended in vivo.

SUBMITTER: Cervantes CF 

PROVIDER: S-EPMC3744861 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature