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Globular proteins: myoglobin


Myoglobin is an example of a monomeric globular protein that can bind oxygen. It is found in muscle tissue of mammals and gets the oxygen from hemoglobin, another oxygen-binding protein, that transports oxygen from the lungs to the muscle tissue. This example will address:
  • secondary structural elements of proteins and their folding into a tertiary structure;
  • distribution of charged and hydrophobic residues in the tertiary structure, solubility of proteins;
  • folding and unfolding of proteins;
  • the existence of prosthetic groups in proteins;
  • equilibrium binding of ligands to proteins, binding constants, on- and offrates;
  • differences between myoglobin and hemoglobin, e.g. quaternary structure, affinity, cooperativity of binding (hemoglobin)
Proposed book chapters: NC 4, 5.1and VV 8, 9, 10.

Reset Pymol
myoglobin
Image by David Goodsell from the "Molecule of the Month" section of the PDB.
Myoglobin was the first protein to be structurally characterized. It was in 1957, in the laboratory of John Kendrew. Myoglobin is a small protein that is present in muscle cells. Its functions is to store oxygen, necessary for muscles to work.

In order to explore its structure, which ultimately leads to understanding its function, first we need to obtain the structure from the PDB repository. There, if we search for "myoglobin" we obtain around 230 structures. Making use of the utilities on the left side of the page, we can "Sort Results" -> by "Release Date". We will work with the first structure, 1mbn, in this tutorial.

Without leaving the PDB site, we can learn a lot about this particular structure: aspects related to the experimental procedure that was followed to resolve it, its classification, its composition, its function and a comprehensive description of its geometric parameters.
Launch Pymol

Load 1mbn
To start interacting with the structure, launch pymol (Instructions to configure Firefox to interact with PyMOL) and wait until the word "Ready" appears.

NOTE: Observe that in Windows, once a window get the focus, it comes to the front, preventing you to see the rest of the programs you are working with. Everytime you click one of the links, something is happening in PyMOL. Try to adjust the size of your windows so that you see PyMOL and Firefox both at the same time.

Then click to load the structure into Pymol. The 'heme' group is clearly distinguishable from the rest of the protein. In a standard use of Pymol, users download the pdb file from the PDB database to their computers. To load the structure in the program use the "Open" option in the "File" menu (top left).
Show surface
show surface






Show heme
set stick_ball
set stick_radius, 0.12
set stick_ball_ratio, 1.5
unbond name fe, name N*
unbond name fe, name O*
set sphere_scale, 0.25
show sphere, name fe
show sphere, resn OH and name O
label resn OH and name O, "  H2O"
label resn hem and name fe, "  %s" % name
show sticks, resn hem
color iron, name fe
color nitrogen, resn hem and name N*
color carbon, resn hem and name C*
color oxygen, resn hem and name O*
create heme, resn hem
One can get a general overview of a structure showing a "surface display". Click on the link and use the mouse to rotate (left click) the structure. Try to find the heme group and observe how nicely it fits into the pocket where it is located. You can also zoom (in or out) by moving the mouse forward and backward while maintaining the right button of the mouse pressed.

The "Show heme" link, displays more clearly the heme group, removing the wrongly guessed covalent bond between the iron and the nitrogens and the water molecule (note that X-ray crystallography is unable to "see hydrogens", and thus they are generally not represented in the structures). To explore more easily this prosthetic group, click on the name of the protein object 1MBN to hide it for a while. To display it again, just click a second time.

Let's now use the menu associated to the object (menu explaination here) The surface of a protein can be also displayed by clicking on the "S"(how) next to the name of the object of interest (1MBN in our case) and then selecting "surface". Try to do it by hiding it first, using the "H"(ide) option, next to the "S". As you see, under both S and H there are more options, such as "lines" (standard view), "sticks", "ribbons", "cartoons". This different representations are used to better understand the structure of a protein, by means of schematic representations. Try for example the "cartoons" representation to display the secondary structure elements (remember to hide the surface first!)



Color by residue
color whiteselect neg, resn asp+glucolor red, negselect pos, resn lys+argcolor blue, posselect his, resn hiscolor green, his
To start getting a feeling of PyMOL capabilities, and an overview of some chemical aspects of this molecule, let's do some basic operations. First color all the protein white (in the names panel, under "C" menu, select white). Then color the negatively charged aminoacids (ASP and GLU) red. Color in blue the positively charged aminoacids (LYS and ARG). And finally, in green HIS. Observe the distribution of the colors all around the surface. Are there any patterns? Try going back to the surface representation.

Try to use other colors. To know how many colors are available, in the command line type "color" and the press the TAB key.







Display by hydrophobicity


hide all
show cartoon,all

select hydrophobes,(resn ala+gly+val+ile+leu+phe+met)
show sticks, (hydrophobes and (!name c+n+o))
show sticks, (not hydrophobes and (!name c+n+o))
color orange,hydrophobes
color palecyan, not hydrophobes and not resn hem
disable hydrophobes
From that view it is clear that the content of alpha-helices is very high (around 75% of the protein). In total there are 8 right handed alpha-helices. They are connected by short non helical regions. To see this even more clearly, on the "C"(olor) menu, select by ss (secondary structure).

Myoglobin is a very soluble protein, and that property can be easily understood if one looks at how hydrophobic and hydrophilic side chains of the aminoacids are distributed in the structure. Just go around any of the helices and observe how orange appears preferently towards the interior of the protein.













Show binding site in sticks
To conclude, let's explore more thoroughly the binding site and the heme group. First, try to identify the residues that form the binding site, by clicking on them with the mouse. Create a new object constituting the binding site. For that, left-click on any atom of the desired residue. A new selection will be created, named "sele". To add residues to this selection, left-click on any atom of the following residue. To remove a residue, click again on it. Rotate the protein to identify the following residue.

Once that the selection has been created, rename it ("A"ctions-> "rename selection") to a more meaningful name. Label the residues ("L"abel -> residues).

Show all the protein in "lines" and the binding site in sticks.

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