Structural Bioinformatics Lab

Aims

• To give practice in using a molecular graphics program.
• To reinforce the concepts of sequence similarity, structure similarity and the evolutionary relationships between protein molecules.

Objectives

After this exercise you will:

• be able to compare a pair of protein structures using the Swiss PDB Viewer;
• be aware of the degree of structural similarity that is exhibited by pairs of proteins that share an evolutionary ancestor.

The Swiss PDB Viewer

In this lab you will use the Swiss-PdbViewer (also known as DeepView). This is a molecular visualisation and modelling program. You will need to download this program, and install it locally on the computer your are using. Click on "Download" from the menu on the left side of the web page, then follow the instructions to download version 4.1.

Structures from the Protein Data Bank

In this lab you will compare the structures of several haemoglobin structures (from human, horse and marine bloodworm), and also the structure of leghaemoglobin from the root nodules of the plant yellow lupine.

Download the following structure files (local access only):

• Human haemoglobin, alpha chain
• Human haemoglobin, beta chain
• Horse haemoglobin, alpha chain
• Horse haemoglobin, beta chain
• Marine bloodworm haemoglobin
• Leghaemoglobin from yellow lupine

Comparing two protein structures

The following sequence of steps will load two protein structures into the Swiss PDB Viewer, remove their side chains from the display (so that the conformations of the proteins' main chains can be seen more easily), colour the two proteins differently (so that their structures can be compared visually more easily), and produce a pairwise alignment.

1. Launch the Swiss PDB Viewer program (spdbv).
2. From the "File" menu, select "Open PDB File ...", and load the first protein structure file, e.g. human_alpha.pdb.
3. From the "Wind" menu, select "Control Panel".
   1. To remove side chains from the display, click on "side" at the top of one of the columns in the Control Panel.
   2. To make this protein a single colour, press the shift key, then click on one of the boxes in the "col" column. All of the boxes in that column will turn black, and a pop-up window with a palate of colours will be displayed. Choose a colour (e.g. white), and select "OK".
   3. To highlight the heme group, find the row labelled "HEM" at the bottom of the Control Panel, click on the box in the "col" column of this row, choose a contrasting colour for the heme group, and click "OK".
4. Repeat steps 2 and 3 to load a second protein, and colour it differently to the first protein (e.g. red).
5. To superpose one protein onto the other, select "Iterative Magic Fit" from the "Fit" menu.
6. Click on the following symbol to position the superposed structures in the centre of the graphics window:
   

   

7. To see an alignment of the sequences of the two proteins, select "Alignment" from with "Wind" menu.
1. If you place the cursor over a character in the Alignment window, that residue will be highlighted (flashing blue/yellow) in the 3D structure.
2. To see a formatted sequence alignment, click on the text symbol in the upper left of the Alignment window.
3. If you want to save the sequence alignment to a file, go to the "File" menu, then the "Save" submenu and select "Sequence Alignment ...". Note that the lines with stars and dots indicating conserved positions might need to be adjusted to line up with the columns in the alignment.

Questions

1. Compare the alpha chain of human haemoglobin and the beta chain of human heamoglobin.
   1. Describe the degree of sequence similarity and structural similarity between these two protein chains.
   2. Identify in the 3D structure the parts of these two proteins that differ in length (i.e. where there are gaps in the alignment). Describe where these regions are located (e.g. with respect to the alpha-helices, proximity to the heme group, proximity to the centre of the proteins).
2. Compare the alpha chain of human haemoglobin and the alpha chain of horse heamoglobin. Describe the degree of sequence similarity and structural similarity between these two protein chains.
3. Compare the alpha chain of human haemoglobin and marine bloodworm haemoglobin. Describe the degree of sequence similarity and structural similarity between these two protein chains.
4. Compare the alpha chain of human haemoglobin and leghaemoglobin from yellow lupine. Describe the degree of sequence similarity and structural similarity between these two protein chains.
5. What do these comparisons (and possibly some other pairwise comparisons between proteins in this set) indicate about the evolutionary relationships among these proteins?