Hydrodynamic properties of Vibrio harveyi acyl carrier protein and its fatty-acylated derivatives.
The amino acid sequence of Vibrio harveyi acyl carrier protein (ACP) is 86% identical to that of Escherichia coli ACP, 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 that V. harveyi ACP is more hydrophobic than E. coli ACP in the loop region, a prediction supported by stronger binding of V. harveyi acyl-ACPs (C12 to C16) to octyl-Sepharose. Gel filtration experiments indicated that both ACPs undergo a similar conformational expansion when pH was elevated from 7.5 (R(s) = 24 A) to 9.0 (R(s) = 30 A). Fatty acylation reversed this expansion: R(s) for 16:0-ACP was 12 A, independent of ACP source and pH. By contrast, V. harveyi and E. coli ACPs exhibited distinct gel electrophoretic properties. Fatty acylation of V. harveyi ACP produced a greater increase in mobility on a conformationally sensitive native gel system. Moreover, while both V. harveyi and E. coli ACPs 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 that E. coli and V. harveyi ACPs 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.
de la Roche
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Atlantic Research Centre, Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada.
Shen
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Byers
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D000213:Acyl Carrier Protein / Q000031:analogs & derivatives / Q000737:chemistry*; D000215:Acylation; D000595:Amino Acid Sequence; D019730:Carbon-Sulfur Ligases; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008025:Ligases / Q000378:metabolism; D008969:Molecular Sequence Data; D012685:Sepharose / Q000031:analogs & derivatives / Q000378:metabolism; D014733:Vibrio / Q000737:chemistry* / Q000201:enzymology
D000213:Acyl Carrier Protein; D005227:Fatty Acids; C027176:octyl-sepharose CL-4B; D012685:Sepharose; D008025:Ligases; D019730:Carbon-Sulfur Ligases; C018749:long-chain-fatty-acid-(acyl-carrier-protein) ligase