This protocol facilitates the study of aminopeptidases with different oligomeric states and oligomerization-dependent activities that are balanced between inactive dimers and active dodecamers. This conventional technique can be easily performed in any lab. And aside from requiring access to a beamline or x-ray crystallography facility, does not require special devices.
With some adaptation, this method may be applied to any other protein with an activity or function that is dependent on its degree of oligomerization. To process the x-ray data, users must have a minimal understanding of crystallography. We invite users to follow trainings, to visit XDS Wiki, and to watch Phenix tutorials.
To perform an activity assay, add 25 microliters of 100 mM L-Leucine p-Nitroanilide to 965 microliters of 50 millimolar MOPS, 250 micromolar cobalt two chloride and 10%methanol Pre incubate the reaction mix in a 75 degrees Celsius dry bath, before diluting the enzyme to a one micromolar final concentration with additional 50 millimolar MOPS. Next, add 10 microliters of one micromolar enzyme to the reaction mix and vortex the resulting solution before returning the reaction to the 75 degrees Celsius dry bath for no more than one hour. When the solution has turned yellow, stop the reaction with one milliliter of 20%acidic acid and vortex the mixture before allowing it to cool to room temperature.
Then transfer an aliquot of the reaction mix to a spectrophotometer cell and read the absorbents at 410 nanometers against an incubated reaction mix without enzyme, as a negative control. For apoenzyme preparation, add 10 microliters of 1, 10-phenanthroline stock solution to 890 microliters of 50 millimolar MOPS, 0.5 molar ammonium sulfate and 100 microliters of purified TmPep1050. Check the activity loss using the activity assay as demonstrated without the addition of cobalt two chloride.
After the assay, transfer the sample into a dialysis tube and dialyze the sample against 200 milliliters of 50 millimolar MOPS and 0.5 molar ammonium sulfate at four degrees Celsius. Exchange the dialysate with fresh buffer three times during the 48 hour dialysis and collect sample from the dialysis tube. Using ultra filtration units with a 30 kilodalton cutoff, concentrate the sample back to 100 microliters and check the concentration on a nano volume spectrophotometer at 280 nanometers.
To prepare the dimers dilute the apoenzyme to a one micromolar concentration in 50 millimolar MOPS and 0.5 molar ammonium sulfate, and incubate the reaction for two hours in a 75 degrees Celsius dry bath. Then concentrate the enzyme sample to when at least 50 microliter concentration and check the molecular weight by size exclusion chromatography. To improve the shape, size and crystallinity of the protein crystals, add the appropriate volume of optimized crystallization solution to a drop of crystals to bring the crystal droplet volume to 10 microliters.
Transfer the droplet to a 1.5 milliliter micro tube and add 90 more microliters of crystallization solution to the seeded well. For each seed dilution, add 500 microliters of crystallization reagent per well to the appropriate number of wells in a crystallization plate and mix two microliters of protein sample with two microliters of crystallization reagent and 0.2 microliters of seeds in one crystallization support per well. Then fix the supports on to each well of crystallization reagent.
For crystal picking, fill a bath with liquid nitrogen and plunge any vials or basket used for sample handling into the nitrogen. Handling liquid nitrogen is hazardous it can cause severe frostbites. Be sure to wear proper individual protections like cryogenic gloves and protective eye goggles.
Set up sample picking loops of different sizes according to the crystal size and use a binocular to select a drop containing crystals. Then use a loop to gently select an isolated crystal from one well and immediately plunge the loop in liquid nitrogen before placing the loop in a suitable vial. To measure diffraction spot intensities, create a folder from which XDS will be run and locate the path to the images.
To run xdsme, enter the command in a terminal window. After XDS has ended the job check the correct lp file and note the probability of the space group determination, data completeness, the highest resolution, crystal mosaicity and data quality. After checking the XDS pointless log to obtain the likelihood of space groups, use the commands to rerun xdsme with different space group solutions proposed by XDS, in a separate folder to avoid overriding the previous process.
After selecting the best solution based on the data statistics, enter the commands to run the XSCALE and XDSCONV. To determine the phase without an anomalous scattering item, use 4P X, Y coordinates, to prepare the starting model for molecular replacement. From the PDB file, use the Phenix PDB file editor to extract the A monomer and to truncate its amino acids in alanine.
Run X triaged with the reflection file generated by XDSCONV and the sequence as inputs. Check the log file from Xtriage. Note the completeness, the number of subunits in the asymmetric unit, the anisotropy, the presence of ice rings and the twinning occurrence.
To run Phaser-MR in Phenix for molecular replacement, select the reflection file, the sequence and the starting 4P six Y model truncated in polyalanine and click Run. upon completion, check if a model has been found and check the score of the molecular replacement. A translation factor Z score of at least eight, indicates that the solution is definitively correct.
After determining the phase by molecular replacement, select Run Auto Build and click Run. All of the required files will be automatically added. Upon completion, check the model in COOT and build and refine the model manually, according to the electron density map in COOT.
Using this model, the sequence and the diffraction data as inputs, refine the manually curated model in Phenix, referring to Phenix to help to select the right strategy. After refinement check the results, refinement free and refinement work must decrease. MolProbity indicators must be respected and outliers with low real space correlation, must be limited.
When the best refined model has been generated, run MolProbity on the server and check any outliers identified by the program. Size exclusion chromatography can be used to determine the apparent molecular weight of the purified protein. The crystallization condition of the peptide, can be optimized by varying the pH versus the PEG concentration around the condition of the dimer.
For example, the best TmPep1050 H60A H307A crystals, were obtained in 0.1 molar sodium citrate with a pH of 5.2 and 20%PEG 3350 concentration, after one cycle of micro seeding to improve the micro crystallinity. In this analysis, data indexation showed that the space group of the TmPep1050 H60A H307A crystal was C2221, but XDS proposed another solution, the MP space group. The structure of TmPep1050 H60A H307A, could be completed after several cycles of automated and manual refinement.
Confirming the oligomeric state with an interface surface of 1, 710 square angstroms between both monomers and a free energy of interface formation of minus 16.2 kilocalories per mole. Here, a closeup of the TmPep1050 H60A H307A active site compared to the active site of the TmPep1050 dimer and dodecamer can be observed. When building a model take care to build only what you see in the electron density to avoid over interpretation, the data, and to take the time to polish your model.
The molecular weight of the dimer and dodecamer can also be determined by native mass spectrometry. And the methods can be useful for detecting transition oligomers.
