The overall goal of this procedure is to observe the dynamic assembly of protein complexes in different pH environments using biolayer interferometry and confirm the components using electron microscopy and mass spectrometry. Identifying and understanding the specificity governing protein complex formation and assembly is of extreme interest to biochemical researchers. The methodology we're talking about today allows researchers to assemble, visualize and validate predicted macromolecular complexes.
The complex nature of the biosensor surface then allows researchers to easily release assembled complexes into small microvolumes for electron microscopy and mass spectrometry analysis. In this demonstration, we followed the kinetics of pH-induced structural rearrangements of the anthrax toxin complex and verified these rearrangements by electron microscopy and mass spectrometry methods, all while avoiding aggregation. Demonstrating the anthrax toxin assembly on the BLI biosensor surface, macro sample release and EM-negative staining procedures will be Alexandra Machen and Pierce O'Neil, graduate students from my laboratory.
Dr.Antonio Artigues is instrumental in analyzing and identifying the BLI-released microsamples, using the Orbitrap Fusion Lumos mass spectrometer. To begin, hydrate an amine reactive second generation BLI biosensor tip by immersing it in 250 microliters of water and incubate it for 10 minutes. In the Blitz software, program the step times as listed in this table.
Start the BLI run by immersing the biosensor tip in 250 microliters of water for 30 seconds to measure an initial baseline of biosensor thickness and density. Next, activate the biosensor by immersing the tip into 250 milliliters of 50 millimolar NHS and 200 millimolar EDC for seven minutes. Then, immerse the activated biosensor in 50 millimolar PDEA dissolved in 0.1 molar borate buffer, pH 8.5, for five minutes to generate an activated bioreactive surface.
Now, immerse the activated bioreactive biosensor in 250 microliters of a solution containing 100 nanomolar E126C LFn in 10 millimolars sodium acetate pH five, 100 millimolars sodium chloride buffer for five minutes. Immerse the LFn tip in 50 millimolar L-cysteine, 1 molar sodium chloride, 0.1 molar sodium acetate pH five for four minutes to quench any remaining free thiol-reactive groups. After immersing the quenched LFn tip in 50 millimolar Tris, 50 millimolar sodium chloride to establish buffer baseline, immerse the tip into 0.5 micromolar protective antigen prepore, 50 millimolar Tris, 50 millimolar sodium chloride for five minutes to create the LFn PA-prepore complex.
Once PA-prepore is associated, remove the tip from the PA-prepore solution and immerse the tip into 50 millimolar Tris, 50 millimolar sodium chloride for 30 seconds to wash away any non-specifically bound PA-prepore. Immerse the LFn PA-prepore complex into 0.5 micromolar CMG2 in 50 millimolar Tris, 50 millimolar sodium chloride for five minutes. Immerse the LFn PA-prepore CMG2 complex into 50 millimolar Tris, 50 millimolar sodium chloride for 30 seconds to wash away any unbound CMG2 to form a pre-endosomal complex.
For EM analysis, release the LFn PA-prepore CMG2 complex from the biosensor tip by immersing the tip into a PCR tube containing five microliters of 50 millimolar DTT, 50 millimolar Tris and 50 millimolar sodium chloride. For tandem MS analysis of the peptides from the complex, release the LFn PA-prepore CMG2 complex from the biosensor tip by immersing the biosensor into a PCR tube containing five microliters of 50 millimolar DTT, six molar keratin-free guanidine hydrochloride and 25 millimolar ammonium bicarbonate, pH eight. To view the complex after pH transition, assemble the LFn PA-prepore CMG2 complex on the biosensor, as just demonstrated.
Immerse the biosensor tip into 10 millimolar acetate pH five for five minutes to initiate the transition of the PA-prepore to PA-pore. This transition is indicated by increasing amplitude, followed by a larger amplitude decline that is hypothesized to be substantial for complete receptor dissociation due to diminished binding affinity. For EM analysis, immerse the biosensor tip in a solution containing 1.25 millimolar micelles for five minutes, to prevent aggregation in solution after disulfide release.
To carry out negative stain electron microscopy, begin by glow discharging a carbon-coated copper 300 grid at 0.38 millibar stable atmosphere pressure and minus 15 milliamps for 20 seconds, then vent the device with air. Secure the grid between a pair of clean tweezers. Then pipette four microliters of released complex sample onto the grid and allow adsorption for 60 seconds.
With a filter paper wedge, wick away the remaining liquid. Then stain the grid by pipetting five microliters of 0.75%point zero two micron filtered uranyl formate onto it and after five seconds, wick away the excess stain. Allow the grid to dry at room temperature while covered.
Then use the transmission electron microscope to view the sample. Finally, after preparing samples for mass spectrometry according to the text protocol, operate the mass spectrometer using CID and a normalized collision energy of 35 under automatic control to continuously perform one MS scan, followed by as many tandem MS-MS scans as possible in a three second period. The BLI sensogram trace is a real-time read out of the amplitude changes due to the specific addition of the anthrax toxin components.
The first rise is LFn loading onto the tip. After quenching and baseline, PA-prepore binds to LFn, followed by the addition of soluble CMG2, resulting in the assembled pre-endosomal complex. The complex is then subjected to low pH that weakens receptor binding and results in the prepore transitioning to its extended pore confirmation that is confirmed by the addition of micelles.
Shown here are negative stain EM images of complexes upon release from the biosensor. Pre-endosomal sample grids showed densities consistent with intact ternary complexes consisting of LFn PA-prepore CMG2. Post-acidification complex grids show PA transitioned to pore and solubilized by micelle inclusion with no obvious CMG2 density.
As seen by MS, which confirmed the protein complexes, pre-endosomal MS samples contained peptides from all three toxin components with 60.46, 67.97 and 54.15%coverage for LFn, PA and CMG2 respectively. Post-endosomal samples only contained peptides from LFn and PA.The lack of CMG2 is consistent with the observed BLI nanometer decrease during pore formation. The ability to monitor and validate the assembly of large macromolecule complexes is a crucial step towards understanding the specificity and functionality of large biomolecular assemblies.
Our ability to release pre-assembled macromolecular complexes from biosensors into microvolumes for electron microscopy visualization will be extremely useful for structure analysis. Our analysis of the biosensor assembly complexes composition only used 0.04 femtomoles of the release complex to identify its components before and after assimilated endosomal acidification. Biosensor assembly release protocol can be adapted to follow natural but elusive pH-induced endosomal transitions of other bacterial toxins in viral protein structures, with the added bonus of avoiding protein aggregation.
