Abstract
Bacterial acyl carrier protein (ACP) is essential for the synthesis of fatty acids and serves as the major acyl donor for the formation of phospholipids and other lipid products. Acyl-ACP encloses attached fatty acyl groups in a hydrophobic pocket within a four-helix bundle, but must at least partially unfold to present the acyl chain to the active sites of its multiple enzyme partners. To further examine the constraints of ACP structure and function, we have constructed a cyclic version of Vibrio harveyi ACP, using split-intein technology to covalently join its closely apposed N and C termini. Cyclization stabilized ACP in a folded helical conformation as indicated by gel electrophoresis, circular dichroism, fluorescence, and mass spectrometry. Molecular dynamics simulations also indicated overall decreased polypeptide chain mobility in cyclic ACP, although no major conformational rearrangements over a 10-ns period were noted. In vivo complementation assays revealed that cyclic ACP can functionally replace the linear wild-type protein and support growth of an Escherichia coli ACP-null mutant strain. Cyclization of a folding-deficient ACP mutant (F50A) both restored its ability to adopt a folded conformation and enhanced complementation of growth. Our results thus suggest that ACP must be able to adopt a folded conformation for biological activity, and that its function does not require complete unfolding of the protein.
Highlights
Bacterial acyl carrier protein (ACP)2 is a small protein required for the synthesis and transfer
Vibrio harveyi ACP is largely unfolded at neutral pH, but its helical conformation can be stabilized by charge neutralization [13], by binding of divalent cations to helix II [10], or by interaction with partner enzymes [15]
We have recently shown that the L46W mutant of V. harveyi ACP undergoes a pronounced Mg2ϩ-dependent fluorescence blue shift of emission maximum from ϳ355 nm to Ͻ320 nm, reflecting the movement of Trp-46 in helix II into the hydrophobic interior of the folded protein [15]
Summary
Plasmid Construction—PCR reactions were carried out using Phusion DNA Polymerase (Finnzymes) and commercially synthesized primers (Integrated DNA Technologies). For in vitro purification of the linL46W-encoded DNA, the C-terminal portion of GST (starting at an internal MscI-site), and the L46W sequence were amplified using primer pairs GSTC-for/GSTC-rev and LINL46W-for/LINL46W-rev, respectively; products were fused by anneal-extension PCR and subsequently amplified using primers GSTC-for/LINL46W-rev. This GSTC-L46W fragment was cloned into pGEX-L46W using MscI and AfeI, creating pGEX-linL46W. SDS-15% PAGE (160 V, 60 min) and native-20% PAGE (160 V, 90 min) [20] were used to identify fractions containing apoand/or holo-ACPs. To obtain cycL46W or cycF50A protein, BL21(DE3)pLysS cells harboring pTCYC-L46W or pTCYC-F50A were induced, harvested, resuspended in buffer A, and lysed as above. Sausage representation figures for linL46W and cycL46W were created using MOLMOL 2.6.0 [26]
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