A Case Study The Kcsa potassium channel from Streptomyces lividans consists of four identical integral membrane protein subunits 0/27/2005 Chaoqun Wu, Fudan University
10/27/2005 Chaoqun Wu, Fudan University Chaoqun Wu, Fudan University 26 A Case Study The KcsA potassium channel, from Streptomyces lividans, consists of four identical integral membrane protein subunits
Initial protein constructs were obtained by purifying a full-length C-terminal His-Tag protein(170 residues, 18.8 kDa), followed by trypsin treatment to remove the His-Tag Although the trypsin treatment yielded sufficient quantities of protein, this material did not produce diffraction grade crystalS 0/27/2005 Chaoqun Wu, Fudan University
10/27/2005 Chaoqun Wu, Fudan University Chaoqun Wu, Fudan University 27 Initial protein constructs were obtained by purifying a full-length C-terminal His-Tag protein (170 residues; 18.8 kDa), followed by trypsin treatment to remove the His-Tag. Although the trypsin treatment yielded sufficient quantities of protein, this material did not produce diffraction grade crystals
Domain elucidation 1. Detergents(e.g, Mega-9, LDAO)Were found to be suitable for protein purification and crystallization and did not interfere with MALDi-Ms analysis 2. Proteolysis experiments with trypsin chymotrypsin, and subtilisin, then mapped by MALDi-Ms 0/27/2005 Chaoqun Wu, Fudan University
10/27/2005 Chaoqun Wu, Fudan University Chaoqun Wu, Fudan University 28 Domain elucidation 1. Detergents (e.g., Mega-9, LDAO) were found to be suitable for protein purification and crystallization and did not interfere with MALDI-MS analysis 2. Proteolysis experiments with trypsin, chymotrypsin, and subtilisin, then mapped by MALDI-MS
PROTEOLYSIS RESULTS Each protease quickly removed about 45 residues from the C-terminal of the His-Tag KcsA, leaving a 122-residue(by trypsin) or 125-residue(by chymotrypsin or subtilisin) core domain. There was little indication that the 45-residue portion that was proteolytically trimmed from the c-terminal of full length kcs a had any inherent structure Beyond one day of digestion, the C-terminally truncated protein began to show an additional cleavage site 19 residues(by trypsin) or 25 residues by subtilisin) from the N-terminal Chymotrypsin did not cleave anywhere in the N-terminal 0/27/2005 Chaoqun Wu, Fudan University
10/27/2005 Chaoqun Wu, Fudan University Chaoqun Wu, Fudan University 29 PROTEOLYSIS RESULTS: Each protease quickly removed about 45 residues from the C-terminal of the His-Tag KcsA, leaving a 122-residue (by trypsin) or 125-residue (by chymotrypsin or subtilisin) core domain. There was little indication that the 45-residue portion that was proteolytically trimmed from the C-terminal of fulllength KcsA had any inherent structure. Beyond one day of digestion, the C-terminallytruncated protein began to show an additional cleavage site 19 residues (by trypsin) or 25 residues (by subtilisin) from the N-terminal. Chymotrypsin did not cleave anywhere in the N-terminal
Another problem MS revealed that in addition to the expected chymotryptic Cleavage product(KCsA 1-125) Cleavage product (KCSA 1-125), an unanticipated second population of the protein appeared (Kcsa 20-125), not easily distinguished from the expected product by SDS-PAGE, and its presence likely would have adversely affected the crystallization trials 0/27/2005 Chaoqun Wu, Fudan University
10/27/2005 Chaoqun Wu, Fudan University Chaoqun Wu, Fudan University 30 Another problem: MS revealed that in addition to the expected chymotryptic Cleavage product (KcsA 1-125), Cleavage product (KcsA 1-125), an unanticipated second population of the protein appeared (KcsA 20-125) , not easily distinguished from the expected product by SDS-PAGE, and its presence likely would have adversely affected the crystallization trials