DOI: 10.5061/dryad.0rxwdbrzv
title: Type III secretion system effector proteins are mechanically labile

description: Multiple Gram-negative bacteria encode Type III secretion systems (T3SS)
 that allow them to inject effector proteins directly into host cells to
 facilitate colonization. To be secreted, effector proteins must be at
 least partially unfolded to pass through the narrow needle-like channel
 (diameter &lt; 2 nm) of the T3SS. Fusion of effector proteins to
 tightly packed proteins—such as GFP, ubiquitin, or dihydrofolate reductase
 (DHFR)—impairs secretion and results in obstruction of the T3SS. Prior
 observation that unfolding can become rate limiting for secretion has led
 to the model that T3SS effector proteins have low thermodynamic stability,
 facilitating their secretion. Here, we first show that the unfolding free
 energy (ΔG0unfold) of two Salmonella effector proteins, SptP and SopE2,
 are 6.9 and 6.0 kcal/mol respectively, typical for globular proteins and
 similar to published ΔG0unfold for GFP, ubiquitin, and DHFR.
 Next, we mechanically unfolded individual SptP and SopE2 molecules by
 AFM-based force spectroscopy. SptP and SopE2 unfolded at low force
 (Funfold ≤ 17 pN @ 100 nm/s), making them among the most mechanically
 labile proteins studied to date by AFM. Moreover, their mechanical
 compliance is large, as measured by the distance to the transition state
 (Δx‡ = 1.6 and 1.5 nm for SptP and SopE2, respectively). In contrast,
 prior measurements of GFP, ubiquitin, and DHFR show them to be
 mechanically robust (Funfold &gt; 80 pN) and brittle (Δx‡ &lt; 0.4
 nm). These results suggest that effector protein unfolding by T3SS is a
 mechanical process and that mechanical lability facilitates efficient
 effector protein secretion.
description: Circular dichroism data was collected and analyzed for Fig. 1 as
 follows: Protein was removed from the -80 ºC freezer,
 thawed, and then centrifuged at 21,000 rcf for 5 min. A 10 M urea solution
 was deionized using BioRad AG 501-X8 resin (50 g beads/L urea) for 1 h and
 vacuum filtered through a 0.22-μm membrane to remove the resin. Urea
 concentration was measured using an Abbe refractometer. For equilibrium
 unfolding measurements, serial dilutions of urea with a fixed protein
 concentration of 0.05 mg/ml  as well as corresponding no-protein blanks
 were prepared  by mixing 10X SptP buffer (100 mM Tris base, pH 8.0, 1.5 M
 sodium sulfate, and 5 mM TCEP) or 10X SopE2 buffer (250 mM HEPES, pH 7.2,
 1.5 M NaCl, and 5 mM TCEP) with ultrapure water, 10 M urea stock, and
 protein to 1X buffer concentration. Samples were incubated in a 25 ºC
 water bath for 1–3 days to reach equilibrium before CD spectra were
 collected. Measurements were performed using a quartz
 cuvette (Hellma) with a 1-mm path length on an Applied PhotoPhysics
 ChiraScan Plus spectrophotometer. Measurement parameters: λ = 212.5–260
 nm; step size = 0.5 nm; bandwidth = 1.0 nm; time per point = 0.5 s; and 3
 repeats. The instrument was thoroughly purged with nitrogen to prevent
 ozone formation. Temperature was held at 25 °C with a Peltier sample
 holder and the temperature recorded using the temperature probe. Prior to
 loading, samples were spun at 18,000 rcf for 5 min. We measured a control
 sample as “blank” before every protein sample. Following this pair of
 measurements, the cuvette was serially rinsed with several mL each of 10 M
 urea, urea free buffer, 1% cleaning solution (Hellmanex), and ultrapure
 water. The cuvette was then filled with ultrapure water and a CD spectra
 taken to ensure no protein adhered to the cuvette. The cuvette was then
 rinsed with absolute ethanol and dried using filtered house air. This was
 repeated for every concentration of urea. We analyzed
 the CD data using Applied Photophysics software. First, the three
 independent measurements were averaged. The subsequent spectrum was
 smoothed using the Savitzky–Golay algorithm with a window size of 12
 points. This smoothing was done on both the protein-containing sample and
 the blank. We then subtracted the smoothed blank spectrum from the
 smoothed protein-containing spectrum to give the final, baseline corrected
 spectrum. After this analysis was done for all urea concentrations, the
 ellipicity at λ = 222 nm was plotted as a function of urea concentration.
 This plot was fit with an equation to determine the free-energy of
 unfolding assuming a two state system which accounts for a sloping
 baseline. AFM data was collected and analyzed as
 follows for Fig. 2: AFM experiments were performed on a
 Cypher ES (Asylum Research) in a temperature-controlled closed fluidic
 cell (<i>T </i>= 25 ºC). The stiffness (<i>k</i>)
 of the FIB-modified cantilevers was calibrated using the thermal method
 (81) far from the surface while sensitivity was measured by pressing the
 cantilever into hard contact with the surface. The cantilevers had an
 average <i>k ≈ </i>6.5 pN/nm. Force-extension curve
 acquisition was initiated by pressing the cantilever into the surface at
 100 pN for 0–200 ms depending on the surface polyprotein concentration.
 This comparatively low indentation force was enabled by our site-specific,
 cohesin-dockerin-based coupling between the tip and the polyprotein.
 To minimize the compliance
 of the polyprotein construct, we used only a single marker domain and
 short PEG linkers (MW = 600 D), which facilitated detecting proteins that
 unfold at low force and low extension [Fig. 2B,C
 (inset)]<em>.</em> We
 retracted the cantilever at 100–3,200 nm/s while digitizing at 50 kHz. We
 acquired multiple traces per sample by probing the surface in a raster
 scan, moving the AFM tip in a grid pattern with each location separated by
 150 nm. Each spot was probed 10 times unless a molecule was detected, in
 which case the spot was continually sampled until ~20 consecutive attempts
 failed to yield a connection. This meant that an individual protein could
 be repeatedly probed. We found that both
 SopE2<em><sub>CD</sub></em> and SptP<em><sub>CD</sub></em> refolded well, and repeated cycles of unfolding and refolding did not affect the observed unfolding forces (<i>SI Appendix</i>, Fig. S3). The high-bandwidth records were boxcar averaged to the indicated bandwidths for analysis and presentation (1–5 kHz). Force was determined by cantilever deflection accounting for the sensitivity and stiffness of each cantilever. Extension was calculated from the movement of the sample stage minus the deflection of the cantilever. The loading rate (pN/s) for each unfolding event in a force−extension curve was calculated by fitting a line to the force-versus-time curve immediately preceding effector protein unfolding. For the effector protein unfolding-force analysis, only the first unfolding event was used when an unfolding intermediate was observed. A small percentage for the force-extension curves showed atypically high unfolding forces for the initial unfolding of SptP<sub>CD</sub> SopE2<sub>CD</sub> (8 and 2% respectively). These records were excluded from analysis as they most likely represented rare tip-sample surface adhesion and/or unfolding of a misfolded protein.   The data for Fig. S1 was collected as follows: To demonstrate reversibility of urea denaturation, we refolded urea denatured SopE2<sub>CD</sub> and SptP<sub>CD</sub> by dilution. SopE2<sub>CD</sub> was buffered with 25 mM HEPES, pH 7.2, 150 mM NaCl, and 0.5 mM TCEP while SptP<sub>CD</sub> was buffered with 10 mM Tris, pH 8.0, 150 mM sodium sulfate, and 0.5 mM TCEP. Three sample conditions were used, a high urea control sample [4.7 M (SopE2<sub>CD</sub>); 5.6 M (SptP<sub>CD</sub>)], low urea control sample [0.38 M (SopE2<sub>CD</sub>); 0.94 M (SptP<sub>CD</sub>)], and refolded sample (diluted from 4.7 M to 0.38 M for SopE2<sub>CD</sub> and from 5.6 M to 0.94 M for SptP<sub>CD</sub>). Urea buffer without protein was also prepared as a blank. The purpose of the high and low urea controls was to show that the protein denatured under the experimental conditions and to give a signal for native protein for comparison. Samples were prepared and allowed to equilibrate for one day in a 25 ºC water bath. For refolding, the equilibrated samples were added dropwise to a glass vial with a stir bar containing either buffer containing the same amount of urea as the sample (for controls) or buffer with no urea (for the refolding sample). CD spectra were obtained as described in <i>Circular dichroism measurement and analysis</i> in the main text. Two independent high and low urea concentration samples were measured and three independent refolded samples were measured (Fig. S1).   The data for Fig. S3 were collected as follows: Comparisons of the mean <i>F</i><sub>unfold</sub> of the first unfolding event of SptP or SopE2 to the mean <i>F</i><sub>unfold </sub>of the refolded protein. Measurements were collected with a pulling velocity of 1600 nm/s [<i>N</i><sub>first</sub> = 41; <i>N</i><sub>refold</sub> = 12 (SptP); <i>N</i><sub>first</sub> = 18; <i>N</i><sub>refold</sub> = 27 (SopE2)]. Error bars represent the SEM. In the case of an unfolding intermediate, only the first unfolding event of the effector protein was analyzed.
description: Unless noted otherwise, all data and figures were analyzed and
 plotted in Igor.