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MspA is an octameric channel protein in the outer membrane of <jats:italic>Mycobacterium smegmatis<\/jats:italic> and is structurally distinct from any other known outer membrane protein. MspA is the founding member of a family with more than 3000 homologs and is one of the most widely used proteins in nanotechnological applications due to its advantageous pore structure and extraordinary stability. While a conserved C\u2010terminal signal sequence is essential for folding and protein assembly in the outer membrane of Gram\u2010negative bacteria, the molecular determinants of these processes are unknown for MspA. In this study, we show that mutation and deletion of methionine 183 in the highly conserved C\u2010terminus of MspA and mutation of the conserved tryptophan 40 lead to a complete loss of protein in heat extracts of <jats:italic>M. smegmatis<\/jats:italic>. Swapping these residues partially restores the heat stability of MspA indicating that methionine 183 and tryptophan 40 form a conserved sulfur\u2010\u03c0 electron interaction, which stabilizes the MspA monomer. Flow cytometry showed that all MspA mutants are surface\u2010accessible demonstrating that oligomerization and membrane integration in <jats:italic>M. smegmatis<\/jats:italic> are not affected. Thus, the conserved C\u2010terminus of MspA is essential for its thermal stability, but it is not required for protein assembly in its native membrane, indicating that this process is mediated by a mechanism distinct from that in Gram\u2010negative bacteria. These findings will benefit the rational design of MspA\u2010like pores to tailor their properties in current and future applications.<\/jats:p>","DOI":"10.1002\/pro.4912","type":"journal-article","created":{"date-parts":[[2024,2,15]],"date-time":"2024-02-15T13:45:17Z","timestamp":1708004717000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["The C\u2010terminus is essential for the stability of the mycobacterial channel protein <scp>MspA<\/scp>"],"prefix":"10.1002","volume":"33","author":[{"given":"Mikhail","family":"Pavlenok","sequence":"first","affiliation":[{"name":"Department of Microbiology University of Alabama at Birmingham  Birmingham Alabama USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rashmi Ravindran","family":"Nair","sequence":"additional","affiliation":[{"name":"Department of Microbiology University of Alabama at Birmingham  Birmingham Alabama USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"R. 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