Adam B. Shapiro, AstraZeneca R&D Boston, MA; Carl Peters, BMG LABTECH, Cary, NC, 07/2013
Peptidoglycan is an important structural component of the bacterial cell wall. The continual synthesis of peptidoglycan and remodelling of the cell wall is essential for most bacteria. Previously identified antibacterial drugs, such as penicillins, disrupt steps in the synthesis of peptidoglycan and thus cell wall integrity.
Since the enzyme phosho-N-acteylmuramoyl-pentapeptide translocase (MraY) catalyzes one of the last cytoplasmic steps in the biosynthesis, it represents a target for antibacterial drugs. This integral membrane protein catalyzes the attachment of soluble UDP-N-acetylmuramoyl-pentapeptide (UNAM-pp) to the lipid undecaprenyl phosphate (C55P) which is membrane bound.
In this application note we want to present a novel homogeneous FRET-based assay to monitor the activity of MraY. A donor fluorophore (BODIPY-FL) is attached to UNAM-pp (B-UNAM-pp) while the acceptor fluorophore-labelled 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (LRPE), is embedded in micelles which also contain MraY and the lipid substrate C55P (Figure 1).
Fig. 1: Schematic of FRET assay for MraY activity
Micelles containing MraY from E. coli membranes (blue) and C55P (black) are mixed with UNAM-pp (purple) labelled with FRET donor (green). MraY transfers UNAM-pp to C55P bringing the FRET donor into close proximity with the FRET acceptor (red).
MraY translocase activity will attach B-UNAM-pp to C55P bringing FRET acceptor and donor close to one another. Excitation at 485 nm will result in energy transfer, reducing donor fluorescence (at 520 nm) and increasing acceptor fluorescence (590 nm).
Preparation of UNAM-pp and cloning, expression of MraY in E.coli as well as subsequent preparation of membranes from E.coli overexpressing MraY is described in Shapiro et al (1).
A 6 µL mixture containing 0.06% Triton X-100, C55P and E.coli membranes containing MraY was preincubated for 30 min. Addition of 3µL of B-UNAM-pp +/- UMP initiated the reaction. Reaction buffer consisted of (final concentrations): 50 mM Tris-HCl (pH 7.5), 100 mM KCl, 50 mM MgCl2, 1 mM dithiothreitol and 0.05% Triton X-100. Final concentration of reactants: 20 µM C55P, 50 µg/mL membrane protein, 2µM B-UNAM-pp with 0-24 µM LRPE and 0 or 5 mM UMP.
Triplicate MraY assays were performed at room temperature. Fluorescence was excited and simultaneous dual-emission measured using 20 flashes for each reading at a focal height 10.6 mm. Measurements were made every minute for 1 hour and triplicate time courses averaged.
To test the effect of storage at room temperature, MraY assay reagents were stored at room temperature in the dark in the presence or absence of 0.5M trehalose. Reagents were mixed after 0, 2.5 and 5 hours of storage at room temperature and triplicate F590/F520 data were collected at the 1 hour time point. Z’ calculations could then be obtained from samples with (MAX) and without (MIN) membranes.
To study tunicamycin inhibition, reactions contained 16 µM LRPE, 0 or 15 µg/mL MraY-containing E.coli membrane protein, 0.1 µM B-UNAM-pp and 10 µM C55P in assay buffer containing 0.5 M trehalose. Tunicamycin was diluted with assay buffer and average progress curves without membranes were subtracted from the average progress curves with membranes to obtain Δ(F590/F520) and ΔF520 measurements. Percentage inhibition and IC50s were calculated as described in Shapiro et al (1).
When the reaction is exposed to fluorescent excitation at 485 nm a time-dependent decrease in 520 and increase in 590 emission is observed (data not shown). Ratiometric measurements were used as they have less noise than individual fluorescence intensity measurements (Figure 2). This is due to the elimination of fluctuations that equally affect both measurements. The ratio change exhibits an increase in a time and LRPE dependent manner.
Fig. 2: Ratiometric fluorescence measurement as a function of time and LRPE concentration
Δ(F590/F520) is the difference between the 590 nm/520 nm fluorescence emission ratios of reactions without UMP and reactions with UMP.
When reactions are performed in presence of 5 mM UMP the change in fluorescence ratio is negligible (data not shown). In the absence of UMP a large increase in fluorescence ratio is observed with time (Fig. 2). This effect indicates that the signal observed in the assay is due to the MraY reaction.
The effect of trehalose, a sugar stabilizer (2), on storing the reagents at room temperature was also assessed. Figure 3 shows that Z’ values decrease when assays are stored at room temperature for as little as 2.5 hours. However the addition of 0.5 M trehalose blocks this effect.
Fig. 3: Z' as a function of reagent storage time and stabilizer
Assay reagents were stored at room temperature in the dark for the indicated times. Z’ was calculated from data collected after 60 minutes using reactions with (MAX) or without (MIN) membranes.
Fig. 4: Inhibition of MraY by Tunicamycin
Comparison of IC50 versus reaction times measured with Δ(590/520) or Δ(520).
The sensitivity of this assay to inhibition was studied using tunicamycin (Figure 4). The results show that IC50s measured using Δ(F590/F520) are lower and exhibited less of an increase with reaction time than those measured with ΔF520.
This assay has several advantages over those previously reported (3,4). First: it uses no radioisotopes so no special training, handling and disposal are required. Second: it uses a donor fluorophore with an excitation wavelength in the visible rather than UV part of the spectrum. This reduces interference from test compound auto-fluorescence and absorption. Finally: a FRET assay using the ratio of the emission of acceptor and donor fluorescence intensities upon donor excitation yields greater precision.
The use of Δ(F590/F520) over ΔF520 is advantageous due to greater sensitivity to inhibition, resistance to loss of sensitivity to inhibition and a higher signal-to-noise ratio. Furthermore a Z’ of greater than 0.7 indicates that this approach will be suitable for high-throughput screens.