Electrospray ionization mass spectra of acyl carrier protein are insensitive to its solution phase conformation

Abstract

Electrospray ionization mass spectrometry (ESI-MS) can be used to monitor conformational changes of proteins in solution based on the charge state distribution (CSD) of the corresponding gas-phase ions, although relatively few studies of acidic proteins have been reported. Here, we have compared the CSD and solution structure of recombinant Vibrio harveyi acyl carrier protein (rACP), a small acidic protein whose secondary and tertiary structure can be manipulated by pH, fatty acylation, and site-directed mutagenesis. Circular dichroism and intrinsic fluorescence demonstrated that apo-rACP adopts a folded helical conformation in aqueous solution below pH 6 or in 50% acetonitrile/0.1% formic acid, but is unfolded at neutral and basic pH values. A rACP mutant, in which seven conserved acidic residues were replaced with their corresponding neutral amides, was folded over the entire pH range of 5 to 9. However, under the same solvent conditions, both wild type and mutant ACPs exhibited similar CSDs (6 ϩ -9 ϩ species) at all pH values. Covalent attachment of myristic acid to the phosphopantetheine prosthetic group of rACP, which is known to stabilize a folded conformation in solution, also had little influence on its CSD in either positive or negative ion modes. Overall, our results are consistent with ACP as a "natively unfolded" protein in a dynamic conformational equilibrium, which allows access to (de)protonation events during the electrospray process. (J Am Soc Mass Spectrom 2007, 18, 1525-1532

Intein-mediated Cyclization of Bacterial Acyl Carrier Protein Stabilizes Its Folded Conformation but Does Not Abolish Function

“…Apo-linL46W displayed a broad CSD of ions from 6 ϩ to 9 ϩ (maximum at 8 ϩ ) in positive ESI mode (Fig. 5C), which is similar to that observed for wild-type V. harveyi ACP (34). CycL46W, on the other hand, produced a sharp, narrow CSD of predominantly 6 ϩ and 7 ϩ ions (Fig.…”

“…However, of particular interest here is the difference in charge state distribution (CSD) of cycL46W and linL46W. We recently reported that V. harveyi ACP exhibits a similar broad CSD regardless of its conformation in solution, as manipulated by charge neutralization or fatty acylation (34). We further speculated that ACP may unfold rapidly upon entering the gas phase during ionization, unlike many other proteins (e.g.…”

Capillary electrophoresis–mass spectrometry of intact basic proteins using Polybrene–dextran sulfate–Polybrene-coated capillaries: System optimization and performance

“…So, the acetylation did not cause unfolding of the protein, and did not affect the average charge state of the protein in the gas phase [38,39]. Closer inspection of the EIEs shows a consistent sub-peak in front of the main peaks.…”

Conformational effects in protein electrospray‐ionization mass spectrometry

“…Conformational effects are, thus, at the basis of the most dramatic changes in CSDs and are quite universal. There is only one reported case, Vibrio harveyi acyl carrier protein, showing no spectral changes under conditions known to modulate protein conformational properties in solution (Murphy, Rowland, & Byers, 2007). Further experiments will be needed to explain the observed behavior.…”