Degradation signals in the lysine–asparagine sequence space

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Abstract
The N‐degrons, a set of degradation signals recognized by the N‐end rule pathway, comprise a protein‘s destabilizing N‐terminal residue and an internal lysine residue. We show that the strength of an N‐degron can be markedly increased, without loss of specificity, through the addition of lysine residues. A nearly exhaustive screen was carried out for N‐degrons in the lysine (K)–asparagine (N) sequence space of the 14‐residue peptides containing either K or N (16 384 different sequences). Of these sequences, 68 were found to function as N‐degrons, and three of them were at least as active and specific as any of the previously known N‐degrons. All 68 K/N‐based N‐degrons lacked the lysine at position 2, and all three of the strongest N‐degrons contained lysines at positions 3 and 15. The results support a model of the targeting mechanism in which the binding of the E3–E2 complex to the substrate's destabilizing N‐terminal residue is followed by a stochastic search for a sterically suitable lysine residue. Our strategy of screening a small library that encompasses the entire sequence space of two amino acids should be of use in many settings, including studies of protein targeting and folding.

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