Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding

The overall goal of the following experiment is to evaluate the dynamic structural preferences of the V three crown peptide sequence of the R two strain of HIV via AR folding, and to correlate the results with known neutralization sensitivities of the R two strain. This is achieved by selecting an appropriate fragment of the R two V three loop crown to fold AR bonni. As a second step, the simulation of the folding is performed, which searches all of the possible confirmations of the R two crown fragment and records, the most likely confirmations in a stack file.

Next, the recorded confirmations are analyzed to determine the dynamic structural preference of the R two V three crown that might explain its neutralization sensitivity. Results are obtained that share a preference for a rigid beta strand positions 12 to 14 of the V three loop based on secondary structure, preference and energy distribution of the stack of searched confirmations. Hello, I'm Timothy Cardozo, speaking to you from my laboratory in the Department of Pharmacology at New York University School of Medicine.

Today we will show you a procedure for abio protein folding of a flexible loop on an immunogenic HIV one viral protein known as GP one 20. The flexible loop is known as the V three loop of this surface envelope glycoprotein of the HIV one virus and its tip is known as the crown of the V three loop. This is the region we will be folding.

We use this procedure in our laboratory to help us identify productive pathways towards successful HIV vaccine designs. So let's get started. To begin this procedure, select the V three crown sequence to be folded in silico.

Previous studies from this lab indicate that positions 10 to 22 of any V three loop sequence, give the best results, run the armenio folding experiments with the user friendly pull down menus in the graphical user interface or GUI of the ICM Pro Molecular modeling software. First, the three dimensional atomic structure of the peptide corresponding to this sequence must be built into the computer's virtual space ace. To build the three dimensional atomic structure, go under the file menu and select new.

This will reveal a screen with several tabs. Select the peptide tab and paste or type the sequence into the text box. Click okay to build the three dimensional structure of the peptide.

The structure will appear in ICMs graphical window. Once the three dimensional structure has been built, go to the molecular mechanics menu and select minimize, which will pop up as a side menu. From the side menu, choose global.

This will reveal a screen with several entry fields and check boxes already selected to default parameters. If necessary, change the selection to choose the peptide to be folded. Adjust the length and precision of the simulation by changing the number of global moves and the number of local min calls respectively.

Select all for all atom folding of the peptide. Then select save movie to make a movie file of the folding. Finally, click apply to start the folding in the graphical window.

The algorithm begins to fold the peptide into different confirmations, calculating and recording the peptide energy for each confirmation. Once completed, the most energetically stable confirmation of the peptide as well as alternative confirmations with nearly the same energy will be identified and visualized on the computer Performing peptide folding. Using the gooey demonstrates what folding looks like, but does not allow ideal parameter selection for folding of the V three loop crown.

For this purpose, it is best to perform the folding from the non graphical command line Using a script, A script is simply a series of text commands saved line by line into a document or text file that are fed into the ICM program automatically and executed one after the other to fold the V three loop using a script of command lines. First, write a text file to be saved onto the computer's hard disk in a local directory. As before, begin by building the peptide in the computer's virtual space.

Then give a name to the simulation and set the number of free variables, which are the chemical bonds in the peptide left free to rotate in the folding. Next, specify how long the simulation will run for optimal folding of the V three loop. This will depend on the number of free variables identified in the previous step to determine the precision of confirmational search in the experiment, set the number of search steps to be performed in each local minimum.

Then set other parameters that have been optimized for the simulation based on previous research, including the temperature minimization, gradient, and probability distribution. Following the setting of experimental parameters indicate which energy calculations will be used during the folding. Here, Vander Vi's energy is indicated by VW internal peptide.

Energy is indicated by 14 hydrogen bonding energy is indicated by HB electrostatic. Energy is indicated by El Salvation. Energy is denoted by SF and entropy is denoted by EN.Specify the final settings, including the preferred backbone and side chain angles to be searched and the starting confirmation.

Finally write the command to run and save the calculation. Once the script has been written and saved as a text file, run it from the computer's operating system command loan prompt. As before the most energetically stable confirmation of the peptide as well as alternative confirmations with comparable energy will be identified and saved in the project file for visualization on the computer.

Once the calculation has completed, open the file by selecting file open from the EE pull down menu. Display the molecule by clicking on the checkbox next to it in the workspace panel, select molecular mechanics stack view. To view the energy ranked stack of top peptide confirmations from the folding, click on the plotting tool in the lower right corner of the stack panel to plot the results.

First click okay in the resulting window and then click on the middle tab of the plot called best confo. Then click on the first row of the stack table of results, which is the lowest energy confirmation found in the search. The peptide structure will rearrange into its lowest energy confirmation in the graphical window.

Analyze this confirmation for beta strand like or alpha helical characteristics, especially in the first five positions of the peptide. By selecting this region in the sequence and clicking on the stick icon in the upper left of the screen to display all the atoms. Next, the stack of energy results must be analyzed to plot the best confirmations.

If the lowest energy confirmation is separated by a significant gap from the other confirmations, it indicates a tendency towards a rigid structure. To evaluate the results, open the project vial and choose molecular mechanics stack view a table of the stack confirmations will appear. Visualize the stack confirmations by clicking on the plot histogram icon.

Finally, click molecular mechanics stack play to make a movie at the stack and visualize the confirmational preferences uncovered by the folding here. The results for the R two folding are shown. The confirmation is not alpha helical and is a random coil as expected for V three loop crowns, notably in the fragment at residue 12 through 14.

In the V three loop, a clear beta strand confirmational preference is seen throughout the stack and very few alpha helical confirmations are observed. A local beta strand confirmation is recognized by its extended linear shape. This is the location of the unusual isoleucine proline methionine sequence of the R two strain, a rare sequence in HIV strains at this position, and one that has been hypothesized to be responsible for the unusual features of R two.

Furthermore, an energy gap of almost three units is seen between the lowest energy confirmation and the second lowest energy confirmation. Thus, the structure only flickers out the lowest energy confirmation less than 1%of the time, suggesting that the R two V three crown and its local beta strain confirmation at positions 12 to 14 has a more rigid structure rather than being completely flexible in nature. We've just shown you how to fold a V three loop crown sequence using the abio algorithm implemented in ICM software, and we've shown you how to analyze the results using the R two strain of the HIV virus.

As an example, when doing this procedure, it's important to choose the identity of the folding peptide corresponding to a fragment of the V three crown carefully, and it is also important to interpret the results with regard to confirmational preference and energy using expert consultation from a molecular modeler. So that's it. Thanks for watching and good luck with your experiments.