Nonnatural amino acid incorporation into the methionine 214 position of the metzincin Pseudomonas aeruginosa alkaline protease

Abstract

Background: The alkaline protease from Pseudomonas aeruginosa (AprA) is a member of the metzincin superfamily of metalloendoproteases. A key feature of these proteases is a conserved methionine-containing 1,4-tight beta turn at the base of the active site zinc binding region.

Results: To explore the invariant methionine position in this class of protease, incorporation of a nonnatural fluorinated methionine, L-difluoromethionine (DFM), into this site was accomplished. Although overproduction of the N-terminal catalytic fragment of AprA resulted in protein aggregates which could not be resolved, successful heterologous production of the entire AprA was accomplished in the presence and absence of the nonnatural amino acid. DFM incorporation was found to only slightly alter the enzyme kinetics of AprA. In addition, differential scanning calorimetry indicated no significant alteration in the thermal stability of the modified enzyme.

Conclusion: Although invariant in all metzincin proteases, the methionine 214 position in AprA can be successfully replaced by the nonnatural amino acid DFM resulting in little effect on protein structure and function. This study indicates that the increased size of the methyl group by the introduction of two fluorines is still sufficiently non-sterically demanding, and bodes well for the application of DFM to biophysical studies of protein structure and function in this class of protease.

Figure 1

Ribbon representation of alkaline protease from P. aeruginosa (AprA). The active site zinc ion is shown in blue and the conserved methionine in yellow spacefill just below the zinc. Calcium ions in the C-terminal β-roll domain are green. Figure generated using WebLab Viewer Pro 3.7 using PDB entry 1KAP [15].

Figure 2

SDS-PAGE gel summary of the refolding of mature full-length AprA. S represents the supernatant of the crude lysate, W1 is the solution from the first wash of the inclusion body pellet, RF is the refolded protein and HS is the refolded protein after the high salt wash and DA is the refolded DFM-AprA.

Figure 3

Positive ion mode electrospray mass spectrum of the refolded, full-length, mature AprA. The major peak in the spectrum (49 500 Da) corresponds exactly to the recombinant protein without the initiator methionine. The peak at 49 534 Da may represent a calcium adduct. The peak at 49 632 Da corresponds to AprA retaining the N-terminal methionine.

Figure 4

Positive ion mode electrospray mass spectrum of DFM-AprA. The calculated molecular weight of the labelled protein with the initiating residue was 49 702 Da, and without, 49 535 Da. Possible sodium adducts (2Na⁺) are seen for both species at 49 747 Da and 49 581 Da.

Figure 5

Active site of AprA showing the environment of the conserved methionine (M214). A: The three histidine ligands (labelled) are shown along with the zinc ion (in green). The spacefill representation (B) shows the closely packed arrangement around the methionine residue, as well as its proximity to the catalytic zinc and the three histidine residues. Protons (white) were added to the M214 methyl group for clarity. Based on structure from PDB entry 1KAP [15].

Figure 6

Differential scanning calorimetry data of both the unlabelled AprA and the DFM-incorporated AprA. All scans were performed at 1°C per minute in 20 mM HEPES (pH 7.8) and buffer scans were subtracted from sample runs.

Figure 7

¹⁹F NMR spectra of DFM-labelled AprA. The 564.7 MHz ¹⁹F NMR spectrum of DFM-labelled AprA in D₂O with approximately 40 mM HEPES (A) and with the addition of 2 mM Gd(EDTA)^- (B). Proton-decoupled spectrum of DFM-labelled AprA (C).