This method can help answer key questions about integral membrane proteins such as the oligomeric state, their size, overall shape and low resolution structure in solution. Solution studies of membrane proteins requires stabilizing molecules like detergents. This technique has the advantage that it makes detergents invisible and highlights the signal from the membrane protein.
The implications of this technique extend to many biological and medical research questions because over 1/3 of proteins reside in membranes where they perform many vital cellular functions. Visual demonstration of this method is critical as the deuterium labeling and neutron scattering steps are difficult to learn because very few institutions offer training in these techniques. To begin, determine neutron scattering length densities or SLDs using the web application MULCh, modules for the analysis of contrast variation data.
Open the contrast module by clicking contrast located in the left navigation pane. Provide text for a project title and enter the details below for two components as subunit one and subunit two. Enter the molecular formula for each component in the formula text box.
Then enter the volume in cubic angstroms for each component in the box below volume. Finally, enter the details for buffer components. Click submit to perform the neutron contrast calculations.
A results page is generated that provides a table of scattering length density and contrast match points as well as formulae for determining these parameters at any given percentage of D2O in the buffer. Review the scattering parameters with particular attention to the component contrast match points. Grow E.coli cells harboring an inducible expression vector for the target protein sequence in LB medium to an optical density at 600 nanometers or OD 600 of approximately one.
Adapt E.coli cells to deuterium labeled medium prior to scale up for over expression of a deuterated protein by diluting the LB culture one to 20 in three milliliters of minimal medium. After growing to an OD 600 of approximately one, repeat the one to 20 dilutions using minimal medium containing 50, 75, and 100%D2O. Once adapted, continue growing the culture in a bioreactor to increase the yield of deuterated cell mass.
As the D2O content is increased, growth rates will decrease. Proceed to harvest, lyse and purify cells as described in the text protocol. Next equilibrate a size exclusion column with greater than two column volumes of final exchange buffer using a fast protein liquid chromatography system.
After injecting the sample, run the column in isolate fractions corresponding to the protein of interest. Reserve an aliquot of matched buffer for small angle neutron scattering or SANS background subtraction. To collect SANS data, first load samples and buffers into quartz cells.
After ensuring that the beam shutter is closed, approach the sample environment area and place the quartz cells in the sample changer. Record the sample changer position for each sample cell. Check the area and ensure the beam path is free from obstruction before leaving the sample environment area.
Then open the beam shutter. Execute a table scan for automated data collection. Follow the instructions for instrument operation provided by the neutron scattering scientist.
Use Mantid Plot software and Python script for the reduction of SANS data from a 2D image to a 1D plot. To do so, log in to the remote analysis cluster and execute Mantid plot from the command line. Then open the provided user reduction script and place the corresponding scan numbers or identification for the sample in buffer pairs in the appropriate list.
Execute the script to generate reduced data as a four column text file in the specified location. Right click the appropriate work space and Mantid plot and select plot with errors for an initial examination of the scattering profiles. Download the ATSAS software suite and use PRIMUS for data plotting, buffer scaling and subtraction and Guinier analysis.
Launch the ATSAS, SAS data analysis application and load the reduced data files corresponding to the sample and buffer pair. To scale the buffer properly, select the data range at high Q where both profiles are similar in flat and click the scale button located under the operations tab. Increase the data range to view all points, click subtract to perform this operation.
Perform a Guinier analysis of the buffer subtracted sample data using the analysis tab of the SAS data analysis application. Be sure the proper file is selected in the list and click radius of gyration. An automated attempt to perform a Guinier fit will be provided by clicking on the auto RG button.
Expand the range of data used to include all of the low Q data and begin narrowing the data range by taking away high Q points until the Q max times RG limit is below 1.3. Use the plot of residuals to verify that data are linear in the fit range. Make small adjustments to the fit region and monitor the sensitivity of these values to the range of data used in the fit.
Obtain the probability distribution function P of R in gnome. Now start the distance distribution wizard within the analysis tab. Obtaining a good fit to the data is essential to obtaining a quality model.
Determine Dmax, the maximum interatomic distance within the molecule. Estimate a value for Dmax by unchecking the box to force Rmax equals zero and entering a large value for Dmax. The first X intercept in the plot of P of R yields this estimate.
Make incremental changes to this Dmax value as well as the range of data used for number of points used to optimize the gnome fit to the data and the resulting P of R curve. Start the PRIMUS shape wizard from the dammif button on the analysis tab of SAS data analysis and use a manual selection of parameters. Define the Guinier range from the fit and proceed to the next step using the navigation buttons.
Define fit values from the P of R plot and proceed to the next step. Provide parameters for the ab initio modeling process. Then initiate the process using the commit button.
Overlay and superimpose the final sans envelope with a related high resolution model using supcomb within ATSAS. Place a copy of the high resolution PDB model to be fit to the SANS envelope in the working directory. Then execute supcomb from the command line using the PDB file names as two arguments with the template structure listed first.
Finally visualize the ab initio model results using PyMOL, a 3D molecular graphics viewing program. Once the SANS envelope and high resolution structure have been superimposed, these models can be visualized using any molecular graphics viewing program. An overview of the PyMOL visualization process is shown.
Once the PDB structure files have been opened in PyMOL, the models should be visible in the PyMOL viewer window. The representations of each model can be changed using the S button next to each file name. Here a surface representation is used for the SANS envelope and a cartoon representation is used for the protein backbone of the high resolution model.
A suitable color scheme can be selected from the options available under the C button. For protein chains, a chainbow coloring provides a color gradient from N to C terminus to aid interpretation. Transparency is applied to the surface representation to allow better visualization of the protein structure within the envelope.
For publication images, a white background is recommended. Once these visualization cues have been applied the 3D structures can be examined by clicking and dragging the structure. Manipulations of the perspective and molecule rotation and translation can be performed.
While attempting this procedure it's important to remember to accurately match the neutron contrast of the detergent head and tail groups with the D2O H2O solvent. After its development, this technique paved the way for exploring the structure of intramembrane asparto proteases. These proteins are involved in the immune response, hepatitis C and Alzheimers disease.
Don't forget that working in biochemical laboratories and at neutron beam facilities can be hazardous. Precautions such as wearing personal protective equipment and following all facility safety rules and radiological postings are mandatory while performing this procedure.
