Introduction to Swiss Pdb Viewer:
The structure levels in proteins

Practical 1 within the course 1MB580, "Macromolecular structure and function" (Molecular Biotechnology, Uppsala University 2008)

Goals

The purpose of this computer exercise is to give an introduction to the molecular graphics program Swiss Pdb Viewer and to protein structures and their building blocks. You will learn how to display proteins in different ways, how to select and display different parts of the protein, how to superimpose structures and how to measure distances.
A first look at protein structure-function relationships will be done by looking closer at the oxygen binding proteins myoglobin and hemoglobin.

Lab report


Introduction

Hemoglobin and myoglobin
All globin proteins are closely related both in sequence and in three-dimensional structure. The primary function of the myo- and hemoglobins is to bind oxygen. They both consist of globin fold polypeptide chains with a bound heme group. The heme group is a so-called prosthetic group and is indispensable for the oxygen binding function of the protein. The heme consists of four pyrrole rings joined by methene bridges with an iron atom in the centre.

Myoglobin is the red pigment in muscles. The role of myoglobin is to take up oxygen from the blood and function as a reservoir of oxygen in the muscle cell and other tissues. The skeletal muscle of diving mammals is particularly rich in myoglobin, which serves as a store of oxygen during a dive. The first protein structure ever to be solved was that of sperm whale myoglobin. This was done in 1957 in a now classical work by John Kendrew and his coworkers in Cambridge, UK. At this time, myoglobin from the skeletal muscle of sperm whale was selected for X-ray crystallographic studies because it could be isolated in large amounts and forms excellent crystals. Nowadays most structural studies are performed on recombinant protein.

The PDB and pdb files
Coordinates of biological macromolecules are usually written in as simple text format, the 'pdb-file' format. Protein coordinates are stored in databases with links to other information such as sequences, publications etc. The coordinates of most of the structures solved are collected in a database called the Protein Data Bank: PDB @ RCSB. You will learn more about the PDB in practical 3.


Files

All the files that you need can be downloaded here. 1a6n.pdb
1a6m.pdb
oxymyoglobin.spdbv
helixpacking_bg.spdv
3HHB.pdb

Basics of Swiss-PdbViewer

A user's guide to this program is avaiable at http://www.expasy.ch/spdbv/text/ctrlpanl.htm.
In the graphics window, the protein myoglobin should appear.
You can also open a pdb file using "CTRL + O" (i.e., by pressing the CTRL button at the same time as the letter "O"), or "Open PDB file" in the File menu.

You will use two different windows. The protein you just opened is displayed in the graphics window. On this page; you can find a manual for what can be done using the different buttons in the graphics window. Now try these:
The control Panel is a second very useful window. If it didn't appear when you opened your pdb file, you can find it under the Wind-menu.
This page of the Swiss-PDBViewer manual tells you everything about the control panel. You will see several columns. You should see a list of all the amino acid residues in your protein. Two important things: when an amino acid is couloured red it is selected, when it is black it is not. If you press return on your keyboard, only the selected amino acids will be shown.
Selecting more than one residue - try these different variants. The slab function.
When you center on an atom in the middle of the protein it is difficult to see what you want because the center is in the middle of a forest of atoms. When you use the slab function, you only display a slice of the molecule, i.e. the closer part and the further part are invisible. This will be a very useful function when you examine different proteins during the tutorials.
Primary-Secondary-Tertiary-Quaternary Structure

There are four different levels of structures: primary, secondary, tertiary and quaternary structure.

The primary structure is the sequence of amino acids in the protein or peptide (one-dimensional). As you know by now, each amino acid has both a three- and a one-letter abbreviation. SwissPdbViewer uses the three-letter abbreviations.

Q1. Write down the one-letter abbreviations for amino acids 15, 35, 37, 53, 118, 130, 150, 28, 132 and 136 in the a6n structure. Together they form a word.

The secondary structure is a regular structural unit formed by sequence regions in a protein (from a sequence perspective: two-dimensional). Basically, these are of two types: α-helices and β-sheets. There are also a number of less common structural motifs that sometimes are regarded as secondary structures (e.g., β turns, left-handed helices).

Now you will explore some properties of α-helices.
In the Display menu, you can select or de-select "Show Sidechains even if Backbone is hidden". When selected, you can display sidechains without the backbone visible. When it is not selected, you can display either the backbone atoms (only) or the backbone+sidechain.

Now colour the protein based on atom type (CPK). The atoms and bonds are now coloured so that; greyish = C, red = O and blue = N. Hydrogens are missing (should be coloured cyan). Hydrogens can be added. Back to the Contol Panel. In some cases there is a letter to the left of your amino acids, it can be either an s or an h.

Q2. Click on an "h" and press return. What happens and what do these letters stand for? Note that even if you only click on one h or s, all the amino acids close to this one with the same letter in front gets selected.

Q3. How many alpha-helices and how many beta strands can you find in myoglobin?
In this kind of protein fold, the helices are often named by capital letters starting from helix A in the N-terminal.

Look at the helix with and without the sidechains visible (control panel). Helices have a polarity and the direction, N- to C-terminal, is maybe not obvious. Since there is a polarity, the ends of helices are very often located at the surface of protein molecules.

Q4. Try to find out a way/pattern to determine the direction (N->C) of an α-helix and make a simple drawing that indicates this pattern?
Q5. Try to explain why the ends of α-helices can be polar and draw a helix to show the partial charges at the N- and C-terminal ends, respectively (hint: look at the figure of a peptide bond).

Helices as well as beta-sheets are held together by a network of hydrogen bonds. Possible H-bonds can be computed based on distances and angles between potential donors and acceptors. All these H-bonds are not necessarily present all the time, since proteins can be very dynamic.
Q6. Describe the main-chain hydrogen-bonding pattern in an α-helix in a simple drawing.

Helices can be left-handed or right-handed. To check this, use your hands. Point the thumb towards the C-terminal along the helix-axis, the fingers should then be able to "grab" the helix pointing towards the C-terminal.

Q7. Is this helix left-handed or right-handed?

Tertiary structure is the three-dimensional fold of the protein, or how the secondary structure elements of the protein are packed against each other.

The pdb file you have loaded is a near atomic resolution model of myoglobin with no oxygen bound, deoxymyoglobin.
The heme group and other "hetero-compounds" (atoms that do not form the protein or nucleic acid chain) can be found at the end of the Control Panel after the last protein residue.

Q8. How many atoms does the heme iron coordinate in the deoxy state?
Q9. What residue in the protein coordinates the heme iron?
Ribbon representation of protein structure
Q10. Describe how the secondary structure elements are packed together. Are the same helices close together in sequence and in space?

Myoglobin with oxygen bound - how to superimpose structures

Load the file "1A6M", which is an (near) atomic resolution model of oxymyoglobin (myoglobin with oxygen bound).

Now we will superimpose the two models 1A6N and 1A6M on the heme group. You can switch between the two models (called "layers" in Swiss-PdbViewer) by clicking the file name in the grey field located in the top of the Control Panel. You can also open the dialogue "Layers info" in the "Wind" menu.
Measuring distances

When you go between the two layers, you can see the "breathing" of myoglobin, and the approximate size of the structural differences.
To get an accurate measurment of how much a specific atom moves, do the following. Q11. What distance does the sidechain of His64 move to accomodate oxygen binding? Use the measuring tool from the toolbar to find out.

Space-filling representations
This file contains the oxy structure. Oxygen is coloured red and the rest of the molecule is coloured yellow.
This is a space-filling static model of oxymyoglobin. In such a model only exposed residues are seen on the surface.

Q12. Try to explain in general terms how release and rebinding of oxygen can come about, though a bound oxygen will be buried in the interior of the protein.


Packing of helices.
We are now going to exemplify a mode of helix packing found in α-helical structures by looking at helices in the myoglobin structure. In this file, the helices B and G are displayed. The backbone of helix B is coloured white. Four of the side-chains in this helix are coloured red. The backbone of helix G is coloured cyan and four of the side-chains are coloured yellow. Due to the basic geometry of an α-helix, residues separated by four in the sequence are close together on the helix surface.
Q13. Describe the helix packing between the helices B and G. Estimate the angle between the two helices. Why do you think certain angles are preferred in the close packing of two helices?

Quaternary structure: the way several polypeptide chains together can form a functional complex. Many proteins "work" together in complexes, e.g., hemoglobin with four subunits or the ribosome that is put together by a large number of separate protein chains and RNA molecules.
The role of hemoglobin is to transport oxygen from the lungs to the tissues. Oxygen is bound to hemoglobin in the lungs and transported to the tissues where it is released. Whereas myoglobin is a monomer, hemoglobin is a tetramer composed of two copies of two different polypeptide chains, called the alpha- and beta-chains. Human have genes for several other subunits of which most of them are expressed in fetus. 98% of the hemoglobin in human red blood cells has the alpha/beta chains and this is called hemoglobin A. From both the DNA and the protein sequences of the different globins, including the myoglobin, it has been suggested that all these globins have diverged from a common ancestor. The gene duplication events that should have caused this, happened some 600-800 million years ago (myoglobin vs. hemoglobin) and about 500 million years ago (alpha- and beta-chain).

Oxygen binding to hemoglobin is cooperative, which means that binding of some oxygen enhances binding of additional oxygen. The binding of the first oxygen leads to conformational changes that propagate further through the hemoglobin tetramer to alter oxygen affinity at other sites.

Myoglobin vs. hemoglobin
As the last part of this tutorial, you will compare hemoglobin and myoglobin.
Q14. What can you say about these folds?.
Q15. Describe in a simple drawing how the globin enteties are packed together and how the heme groups are oriented.


Part of this practical was originally written 2003 by: Henrik Hansson; Gunnar Berglund & Evalena Andersson, Uppsala Universitet and edited 2006 by: Mats Sandgren, Uppsala Universitet.
The rest was written 2007-2008 by: Maria Selmer, Uppsala Universitet.