Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

The overall goal of this procedure is the biochemical and structural characterization of a carbohydrate substrate binding protein from streptococcus pneumoniae. This method can help provide key data to aid instructional studies of substrate binding protein such as the identification of the buffer stability profiles, oligomerization states, and atomic structures. The advantages of this procedure is that it provides data that can increase the success rate of crystallization and structure solution for substrate binding proteins in a robust and straightforward way.

First, prepare 48 buffer solutions with a pH range of 4.0 to 9.5, and sodium chloride concentrations ranging from zero to 0.5 molar. Dispense 40 microliters of each solution into a 96-well plate. Obtain a purified solution of the chosen substrate binding protein.

Add five microliters of the SBP solution to each well for a concentration of one to two milligrams per milliliter. Then add five microliters of a 20-times concentrated fluorescent stain to each well. Mix the solutions by pipetting up and down.

Seal the plate with transparent adhesive film. Centrifuge the plate at 112 times G at room temperature for two minutes. Configure a real-time PCR system to ramp from four to 99 degrees Celsius at a rate of three degrees per minute with a 10-second hold time.

Set the system to take fluorescence readings every half degree. Set the fluorescence filter setting. and assign the samples.

Run the experiment and identify the buffer condition with the highest melting temperature. Next, prepare a buffer with the chosen pH and sodium chloride concentration that includes 2.5%by volume of glycerol and a TCEP concentration of 0.5 millimolar. Characterize the purified SBP with analytical size-exclusion chromatography with multi-angle light scattering.

Identify the most stable species suitable for crystallization, and use preparatory SEC to obtain pure fractions of those species. Perform pre-crystallization tests to determine the optimal protein concentration for crystallization. Use a crystallization robotic system to dispense the desired commercial crystallization solutions into the solution reservoirs of an optical 96-well sitting drop plate.

Then use the system to dispense 100 nanoliters of the protein solution into each crystallization drop well. Transfer 100 nanoliters of each crystallization solution from the reservoir wells to the corresponding well drops. Seal the plate with optical adhesive film.

Use a microscope or an automated imaging system to evaluate the crystal formation and growth every one to two days for two weeks, and once a week thereafter. When sufficiently large and well-shaped crystals have formed, prepare a batch of the crystallization solution with an additional 25%by volume of glycerol to serve as a cryoprotectant. Next, fill a foam dewar with liquid nitrogen.

Immerse a unipuck sample enclosure in the liquid nitrogen and allow the enclosure to cool. Then cut out the film over a drop containing a crystal of interest. Deposit 0.5 microliters of the cryoprotectant on a cover slide close to the drop or in the empty drop chamber of the same condition if available.

Attach to a magnetic wand a cryoloop mounted on a SPINE standard base. Use the cryoloop to transfer the crystal of interest to the drop of cryoprotectant. Then use the cryoloop to transfer the crystal from the cryoprotectant to the first empty position of the submerged unipuck sample enclosure.

Repeat this process to harvest additional crystals. Once all crystals of interest have been loaded into the sample enclosure, use the puck wand to place the unipuck base plate on the enclosure. Transport the unipuck in liquid nitrogen to the beamline.

Use cryo tongs and a puck dewar loading tool to load the unipuck into the beamline sample changer dewar and to detach the sample enclosure. Then select the sample in the beamline software to load and automatically center the sample in the x-ray beam. Acquire an x-ray fluorescent spectrum and identify any elements that can be used in an anomalous diffraction experiment.

Determine the optimal wavelength for collecting anomalous diffraction data by performing an x-ray absorption edge energy scan. Then acquire three x-ray diffraction patterns at 45 degree intervals in standard oscillation mode to automatically determine the crystal unit cell parameters, symmetry, and diffraction limit. Import the recommended data collection parameters into the beamline software and run a single or multiwavelength anomalous diffraction experiment as appropriate.

The stability of the substrate binding protein SP0092 in various salt buffers was evaluated to identify the optimal buffer solution. The highest melting temperatures were found for buffers with a pH of 6.5 and sodium chloride concentrations ranging from zero to 0.2 molar. A buffer with a pH of 6.5 and a sodium chloride concentration of 0.2 molar was selected for the procedure.

The various oligomerization states of SP0092 were identified and separated with SEC-MALS. An analysis of the SEC profile at different protein concentrations indicated that increased protein concentration triggers oligomerization, suggesting that larger oligomers are more stable at high concentrations. Consistent with this, the large oligomers produced crystals in crystallization trials, whereas the monomer did not.

X-ray fluorescence of both native and selenomethionine-labeled SP0092 crystals indicated zinc bound to the protein in both forms. The anomalous signal was maximized by tuning the incident x-ray wavelength to the x-ray absorption edges of either zinc or selenium. A complete anomalous dataset was then obtained, and automated analysis triggered by the presence of an anomalous signal generated initial maps of the protein structures.

Models built into these initial maps were then refined and validated to produce the final models. After watching this video, you should have a good understanding of how to complete a comprehensive structural characterization of a substrate binding protein or of any potential protein target. The implications of this technique extend towards development of new therapeutics for myrococcal disease, as it provides fundamental insights for structure-based design of inhibitors for this class of proteins.

These methods can also be applied to substrate binding proteins from other organisms, and they potentially can be used for any protein type.