Figure 2. Reversed-phase HPLC fractionation of EndoLys-C digests of GLIC: [³H]AziPm primarily photoincorporate into the hydrophobic, transmembrane domain of GLIC.

A) Shown are the sequences of the GLIC fragments produced by enzymatic cleavage with EndoLys-C (specific for Lys). The residue numbering is that used in the crystal structure (PDB:3P50 (10)) and does not include the 3 additional N-terminal residues (GPM) identified by sequence analysis of intact, expressed GLIC. The transmembrane helices M1–M4 are underlined. The Pro residues used to chemically isolate M1, M3, & M4 during sequencing are bolded as are the Trp residues subjected to chemical cleavage to sequence M2. B) Reversed-phase HPLC fractionation of an EndoLys-C digest of [³H]AziPm labeled GLIC. 82% of the recovered ³H eluted in hydrophobic fractions (>75 % organic, fractions 28–38). In gray is the absorbance profile at 215 nm. C) Selected fractions containing ³H were sequenced, with the peptides detected quantitated in picomoles. In fractions 29–33, the only GLIC fragments detected were those containing the transmembrane helices. ND, not detected.