Structural Alignment of Aquaporins

In this section, you will align the aquaporin molecules loaded in VMD using the Multiseq program.

Starting Multiple Sequence Alignment

Now that you have loaded the four proteins in Table 1.2, you will use Multiseq program to align the proteins and analyze their structural and sequence relationships.

Figure: Multiseq Program Window

1

Within the VMD main window, choose the Extensions menu.

2

In the Extensions menu select Analysis $\rightarrow$ MultiSeq.

This is the main Multiseq program window. The rest of the tutorial and exercises will use features from this window, unless specified otherwise. You may be asked to update some databases if this is the first time you use Multiseq. If this is the case, simply click Yes and wait for Multiseq to start.

By default, Multiseq will align all four loaded molecules, unless you delete the molecule(s) in the VMD Main window. However, note that some crystal structures come with water and detergent molecules, which should not be used to align the structures. In your MultiSeq window, you will find that all the protein structures are categorized as VMD Protein Structures and other molecules, e.g. water, are in the VMD Nucleic Structures category. Keep the four protein structures and delete the two structures under VMD Nucleic Structures by clicking them and press the Delete button on your keyboard.

Setting parameters for Multiple Structure Alignments

Multiple Sequence Alignment uses the program STAMP to align protein molecules.

Figure: Stamp Alignment Options

Before you align the molecules, you may want to change the default parameters needed by STAMP.

1

Choose the Tools $\rightarrow$ Stamp Structural Alignment tool. The window shows several parameters that can be set (Figure 10). For this tutorial, you will use the default parameters.

You can look at the STAMP user guide (see box) for information on how to optimize the STAMP parameters to obtain a better alignment.

Aligning the molecules

Now that you have opened the MultiSeq window and made sure the STAMP parameters are correct, you can align the molecules you loaded into VMD.

1

First, delete two molecules listed under VMD Nucleic Structures (1j4n_X and 1lda_X) by clicking on them (they will turn yellow), and delete the them by using the Delete key on your keyboard.

2

In the Stamp Alignment Options window, choose Align the following: All Structures and go to the bottom of the menu and select OK.

Figure: Structural alignment of four aquaporin molecules.

The molecules have been aligned. You can see this both in the OpenGL window (Fig. 11) and in the MultiSeq program window (Fig. 12). Take some time to look at the alignment. Note that your alignment may not immediately resemble Fig. 11. This is because MultiSeq displays the protein in ``NewCartoon'' representation by default. To compare your alignment result with Fig. 11, change the representations for each protein to ``Tube'' in your Graphical Representations window. You could also change the color for each protein.

3

Click on the OpenGL window (Fig. 11). Move your mouse around, rotating the molecules. Do you think this is a good alignment? Look at the top of the molecules: Can you see the pore?

Figure: Sequence Alignment of four aquaporin molecules.

4

Now, click on the Multiseq program window (Fig. 12). Move the left-to-right scroll at the bottom of the window, and take a look at the residues in the Sequence Display. Observe that there are gaps, represented by dashes, in the sequences when they are aligned.

In the next section, you will start using Multiple Sequence Alignment features for the alignment you just made.