Identification of a key residue in the conformational stability of acyl carrier protein.
Conformational flexibility of acyl carrier protein (ACP) is important for its ability to interact with multiple enzymes in bacterial fatty acid metabolism. We have recently shown that, unlike the prototypical ACP from Escherichia coli, the more acidic Vibrio harveyi ACP is largely unfolded at physiological pH. Mutations D18K, A75H and A75H/D18K were made in recombinant V. harveyi ACP (rACP) to determine the importance of basic residues Lys-18 and His-75 in maintaining the native conformation of E. coli ACP. Both D18K and A75H ACPs were fatty acylated by acyl-ACP synthetase, showing that neither mutation grossly alters tertiary structure. Circular dichroism (CD) indicated that rACP refolded upon addition of MgCl(2) at 100-fold lower concentrations (<1 mM) than KCl, suggesting that divalent cations stabilize rACP by interaction at specific sites. Surprisingly, mutants A75H and A75H/D18K exhibited native-like conformation in the absence of MgCl(2), while the D18K mutant was comparable to rACP. Moreover, the alpha-helical content of A75H, A75H/D18K and E. coli ACPs was more sensitive than that of rACP or D18K ACP to modification by the histidine-selective reagent diethylpyrocarbonate. Together, these results suggest that the partial positive charge of His-75 may be important in maintaining the conformational stability of E. coli ACP at a neutral pH.
Keating
Mary-Margaret
MM
Atlantic Research Centre, Department of Pediatrics, Dalhousie University, Halifax, NS, Canada B3H 4H7.
Gong
Huansheng
H
Byers
David M
DM
D000213:Acyl Carrier Protein / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014733:Vibrio / Q000378:metabolism
D000213:Acyl Carrier Protein; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate