Combinatorial mutagenesis of N-terminal sequences reveals unexpected and expanded stability determinants of the Escherichia coli N-degron pathway.

Although it is known that residues near the N-terminus can influence protein stability, there has not been a comprehensive effort to document how these penultimate residues impact protein stability in prokaryotes. Here, we utilize combinatorial mutagenesis, cell sorting, and next generation sequencing to perform the deepest sequence coverage screen of the bacterial N-degron pathway of proteolysis. We present nuance and exceptions to the N-terminus (P1) functioning as a primary stability determinant. We reveal stability contributions for P2-P5 motifs, including lowered stability for clustered bulky residues and Gln, and heightened stability for negatively charged residues, Pro, and Gly. We find that P1 Cys can be an N-degron component in a sequence-specific manner. Furthermore, we employ stability-predictive machine learning to identify motifs with unexpected fates. Our work expands the stability determinants of the N-degron pathway with unprecedented granularity, serving as a resource for N-degron identification, N-degron design, and future molecular basis elucidation.