Activation of phosphatidylinositol transfer protein alpha and beta isoforms from inclusion bodies.
Fully active phosphatidylinositol transfer protein (PI-TP) isoforms alpha and beta have been obtained from Escherichia coli inclusion bodies. Folding and activation of PI-TPalpha was achieved in the presence of DiC7:0-phosphatidylcholine-Triton X-114 (PtdCho-TX114) mixed micelles. Replacement of DiC7:0-PtdCho with the natural ligands of PI-TPalpha, i.e. long-chain PtdCho and phosphatidylinositol, did not stimulate activation. Efficient activation of PI-TPalpha required a low temperature (4 degrees C), the presence of dithiothreitol, and was achieved at a relatively high protein concentration (i.e. up to 500 microg ml(-1)). The inclusion bodies yielded 10 mg homogeneous PI-TPalpha per liter of E. coli culture. Conditions for full activation of PI-TPbeta were similar to those for PI-TPalpha except that long-chain PtdCho-TX114 mixed micelles and a very low protein concentration (i.e. 10 microg ml(-1)) were required. In contrast to PI-TPalpha, PI-TPbeta lost its lipid transfer activity within a few days. This inactivation could be prevented by addition of beta-alanine. In summary, despite 94% sequence similarity, PI-TPalpha and PI-TPbeta display a striking difference both in their preference for the PtdCho acyl chain length required for activation, and in their conformational stability after folding.
Bouma
B
B
Department of Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, Netherlands. b.bouma@chem.uu.nl
Westerman
J
J
Dekker
N
N
Gros
P
P
Wirtz
K W
KW
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D003902:Detergents; D004229:Dithiothreitol; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins; D008823:Micelles; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; D011487:Protein Conformation; D017510:Protein Folding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D013696:Temperature
D002352:Carrier Proteins; D003902:Detergents; D008565:Membrane Proteins; D008823:Micelles; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; D020033:Protein Isoforms; C118289:SEC24 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D004229:Dithiothreitol