Molecular properties of short chain acyl thioesters of acyl carrier protein.

J E Cronan

doi:10.1016/s0021-9258(18)34627-1

Abstract

S-Acylated derivatives of the acyl carrier protein (ACP) of Escherichia coli were prepared by a specific chemical reaction. The properties of acetyl-ACP, butyryl-ACP, and hexanoyl-ACP were compared with those of long chain (greater than C8) acyl-ACPs. Acylation of ACP with long chain (greater than or equal to C8) acyl groups stabilizes the protein to alkaline pH-induced hydrodynamic expansion, the degree of stabilization being independent of acyl chain length (Rock, C. O., and Cronan, J. E., Jr. (1979) J. Biol. Chem. 254, 9778-9785). This was not true of the short chain length acyl-ACPs. We report that the C2, C4, and C6 acyl-ACPs showed a lower degree of stabilization (greater hydrodynamic radii) under alkaline conditions than the longer chain lengths. The degree of stabilization (assayed by polyacrylamide gel electrophoresis at pH 9.5) was a direct function of acyl chain length (C8 greater than C6 greater than C4 greater than C2 greater than ACP). Long chain (greater than C10) acyl-ACPs bind tightly to octyl-Sepharose whereas ACP does not bind. The strength of binding is a direct function of acyl chain length for C10 to C16 (Rock, C. O., and Garwin, J. L. (1979) J. Biol. Chem. 254, 7123-7128). In contrast to the result expected if the entire acyl chain was accessible, we report that neither acetyl-ACP nor butyryl-ACP binds to octyl-Sepharose. Moreover, both hexanoyl-ACP and octanoyl-ACP bind less tightly to octyl-Sepharose than expected from extrapolation of the data for the longer chain length acyl-ACPs. Both the pH stabilization data and the hydrophobic chromatographic data are interpreted as indicating the presence of an acyl chain binding site on ACP that binds the first 6 to 8 carbon atoms of the acyl chain.

Highlights

(ACP) of Escherichia coli were prepared by a specific consistent with the finding that acyl-ACPsare more stable to chemical reaction
We report that thCe 2, C4, and C6 acyl-ACPs showed a lower degree of stabilizationunder alkaline conditions than thelonger chain lengths
In contrast to the result expected if the entire acyl chain was acces- Materials-ACP was purified from E . coli K12 as previously sible, we report that neither acetyl-ACP nor butrylACP binds to octyl-Sepharose

Summary

Molecular Properties of Short Chain Acyl Thioesters of Acyl Carrier Protein*

We recently have developed a method to chemically synthesize acyl-ACP molecules that retain native protein structure. This method allows the preparation of short chain acyl-ACP derivatives (9). The initial protein which in turn is linked through a phosphodiester bond to the serine 36 residue of ACP (1).ACP has a high content of a-helix and thea-helical content is reversibly lost at alkaline pH values (3). Recent results from this laboratory indicate pH was 9.0 (freshly dissolved Trizma 9.0) and the separation pH was 9.5. The I9FNMR spectrum of 6,6-difluorotetradecanoylACP shows the two fluorines to be nonequivalent and sub-

RESULTS

Hydrophobic Chromatographic Behaviorof Short Chain

Carbon Number

(5). REFERENCES