Hydrodynamic Properties ofVibrio harveyiAcyl Carrier Protein and Its Fatty-Acylated Derivatives

The amino acid sequence ofVibrio harveyiacyl carrier protein (ACP) is 86% identical to that ofEscherichia coliACP, although five nonconservative amino acid differences are concentrated in the loop region between helices I and II (residues 18–25). We have investigated the influence of these sequence differences on the hydrodynamic properties of the two ACPs and their fatty acylated derivatives. Hydropathy analysis suggests thatV. harveyiACP is more hydrophobic thanE. coliACP in the loop region, a prediction supported by stronger binding ofV. harveyiacyl-ACPs (C12to C16) to octyl-Sepharose. Gel filtration experiments indicated that both ACPs undergo a similar conformational expansion when pH was elevated from 7.5 (Rs= 24 Å) to 9.0 (Rs= 30 Å). Fatty acylation reversed this expansion:Rsfor 16:0-ACP was 12 Å, independent of ACP source and pH. By contrast,V. harveyiandE. coliACPs exhibited distinct gel electrophoretic properties. Fatty acylation ofV. harveyiACP produced a greater increase in mobility on a conformationally sensitive native gel system. Moreover, while bothV. harveyiandE. coliACPs migrated anomalously at 20 kDa on SDS–polyacrylamide gel electrophoresis, they exhibited strikingly different behavior on SDS gels upon acylation with longer chain fatty acids. These results indicate thatE. coliandV. harveyiACPs exhibit similar overall pH- and fatty acid-dependent conformational changes, but gel electrophoresis is more sensitive to structural differences due to variations of hydrophobicity and charge.