HYDRODYNAMIC PROPERTIES OF VIBRIO-HARVEYI ACYL CARRIER PROTEIN AND ITS FATTY-ACYLATED DERIVATIVES

The amino acid sequence of Vibrio harveyi acyl carrier protein (ACP) i s 86% identical to that of Escherichia coli ACP, although five noncons ervative amino acid differences are concentrated in the loop region be tween helices I and II (residues 18-25). We have investigated the infl uence of these sequence differences on the hydrodynamic properties of the two ACPs and their fatty acylated derivatives. Hydropathy analysis suggests that V. harveyi ACP is more hydrophobic than E. coli ACP in the loop region, a prediction supported by stronger binding of V. harv eyi acyl-ACPs (C-12 to C-16) to octyl-Sepharose. Gel filtration experi ments indicated that both ACPs undergo a similar conformational expans ion when pH was elevated from 7.5 (R-s = 24 Angstrom) to 9.0 (R-s = 30 Angstrom). Fatty acylation reversed this expansion: R-s for 16:0-ACP was 12 Angstrom, independent of ACP source and pH. By contrast, V. har veyi and E. coli ACPs exhibited distinct gel electrophoretic propertie s. Fatty acylation of V. harveyi ACP produced a greater increase in mo bility on a conformationally sensitive native gel system. Moreover, wh ile both V. harveyi and E. coli ACPs migrated anomalously at 20 kDa on SDS-polyacrylamide gel electrophoresis, they exhibited strikingly dif ferent behavior on SDS gels upon acylation with longer chain fatty aci ds. These results indicate that E. coli and V. harveyi ACPs exhibit si milar overall pH- and fatty acid-dependent conformational changes, but gel electrophoresis is more sensitive to structural differences due t o variations of hydrophobicity and charge. (C) 1997 Academic Press.