Computational protocol: A gating motif in the translocation channel sets the hydrophobicity threshold for signal sequence function

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[…] Eukaryotic Sec61 sequences were retrieved from the National Center for Biotechnology Information (NCBI) database by searching for Sec61 and SecY. After sequence alignment, duplicate sequences or very closely related sequences from a single species were eliminated to obtain a single Sec61 sequence for each eukaryote. Chloroplast SecY sequences and fungal Ssh1 sequences were excluded from the eukaryotic Sec61 alignment. Eubacterial and archaebacterial SecY sequences were retrieved from the NCBI database by searching for SecY and sequence lengths of 350 to 550 residues. From the obtained sequences, we removed duplicate sequences, severely truncated sequences, and a small number of sequences derived from unclassified eubacteria. SecY2 proteins, which have few in vivo substrates, were also removed from the eubacterial alignment. Alignment of sequences was achieved using MUSCLE, and the eubacterial sequences were sorted based on taxonomy. Sequence alignment and analysis were performed using the following software packages: Jalview, BioEdit sequence alignment editor (version 7.1.3), and BioPython (). Sequence logos were made using WebLogo.The hydrophobicity of the signal sequence of CPY and derivatives thereof was estimated using an in vivo hydrophobicity scale () as implemented using the ΔG server (http://dgpred.cbr.su.se/). A window corresponding to residues 1–20 of CPY or its derivatives was used to apply a similar calculation method for CPY deletion mutants and point mutants. The position-specific hydrophobicity of amino acid substitutions at T87, Q129, and N302 was also calculated using the ΔG server and the amino sequences of TM2, TM3, and TM7 of S. cerevisiae Sec61. […]

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