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bioscience⏐explained
Vol 2 ⏐ No 2
Anders Hansson
A closer look at proteins!
The compact version
The first lesson
Introduction ----------------------------------------Download -------------------------------------------RasWin----------------------------------------------To study protein structures with RasWin ---------Major features of proteins -------------------------Summary --------------------------------------------
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Introduction
Proteins
Proteins are molecules with many important functions.
They act as enzymes, hormones, signals on the cellsurface, receivers and transmitters of signals, mechanical support elements, ion channels, transporters and
“motors”. Without proteins, there is no life, as we view
life today. Proteins are normally formed according to
the central dogma: stating that the genetic information
flows from DNA to RNA and further on to proteins.
Here, we will examine the structure of a few of the millions of proteins that exist. The purpose is to illustrate
some general concepts. We will make use of a graphical
program; RasWin (or RasMol), and structure databases,
both made available by the efforts of researchers. To
fully appreciate the lesson, it is necessary to have some
knowledge about amino acids and the structures of proteins..
CORRESPONDENCE TO
Anders Hansson
Rudbecksskolan, Sollentuna
Sweden
[email protected]
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bioscience⏐explained
Vol 2 ⏐ No 2
Protein structure
Proteins are composed of one or several chains of amino acid, which fold into specific secondary structures.
Three such structures we will examine closer are alphahelix, beta-pleated-sheet and loop. These structures
position important amino acids in space, enabling their
side chains to carry out the functions of the protein. Alpha-helices and beta-sheets are held together by hydrogen bonds between the peptide bonds. The program
RasWin (or RasMol) is a tool to view and analyse the
structure of proteins: mainly secondary structures, but
also amino acids in active sites.
The polarity of sidechains
A basic variable in the formation of protein structures is
polarity, often expressed with the terms polar-nonpolar, hydrophobic-hydrophilic, water soluble-soluble in
organic solvents or lipophilic-lipophobic. The side chains
of the twenty common amino acids have highly variable
polarities. In RasWin, amino acids with water soluble/polar side chains are named ”polar”, whereas hydrophobic/non-polar side chains belong to ”hydrophobic” amino acids. Non-polar amino acids are often localized close to each other, binding through hydrophobic
effects. In water-soluble proteins, it is common that the
external amino acid side chains are polar, with the interior of the protein is dominated by non-polar amino acid
side chains. There are also other binding forces between the different parts of proteins; van der Waals
forces, hydrogen bonding, ionic interactions and disulfide bridges all contribute to the various structures of
proteins.
Structural analysis
The three-dimensional arrangements of atoms in proteins are determined by the different techniques,
mainly nuclear magnetic resonance (NMR) and X-ray
crystallography. With NMR, it is possible to obtain a
view of the protein in solution, whereas crystallography
displays the structure in a crystal form. Researchers
deposit their data containing atomic coordinates to publicly accessible databases. With the help of specialised
programes an interactive visual representation can be
obtained. Here we will use the freeware-program
RasWin (or RasMol) to investigate the structure of some
selected proteins. Some of the special functions in
RasWin (or RasMol) will be displayed, and certain particularly important features of protein structure will be
exemplified and discussed in more detail.
Download
Running this material requires downloading of nine files
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bioscience⏐explained
Vol 2 ⏐ No 2
from the Internet; the RasWin .exe- and .hlp-files and
.pdb-datafiles for lysozyme, glucagon, triose phosphate
isomerase, immunoglobulin G, myglobin and two variants of trypsin containing atomic coordinates of proteins.
On the Internet, use the address
http://www.umass.edu/microbio/rasmol/getras.htm
and simply follow the instructions to download RasWin
2.6 and rashlp.exe. Save them together under a suitable folder (for example RasMol).
In the same folder you should also load the following files with atomic coordinates:
• 1IY4 is saved as Lysozyme (NMR-data from human
enzyme)
• 1GCN is saved as Glucagon (peptide from pig).
• 1YPI is saved as Triose phosphate isomerase (this
enzyme is from baker´s yeast).
• 1K6Q is saved as IgG Fab-fragment (parts of a
mouse protein)
• 1L2K is saved as Myoglobin (spermaceti whale material, and neutron diffraction)
• 1HJ8 is saved as Trypsin with benzamidine inhibitor
(salmon enzyme with low-molecular protease inhibitor).
• 3TGI is saved as Trypsin with pancreatic inhibitor (a
test tube mix of rat and cow proteins).
The procedure for downloading is as follows::
• enter http://www.rcsb.org/pdb/ and write the IDnumber; for example 1IY4, mark PDB ID and click
"search"
• click “Download/Display File” and under Download
the Structure File/compression none click on “X” for
PDB
• right-click the molecule, followed by file and save as,
go to your RasMol-folder and save as the specified
name, in this case Lysozyme.
Since these files are all in the public domain, they can
be downloaded and distributed to students via CD discs
or a network in advance. Other protein data files can be
found using the Entrez-site at
http://www.ncbi.nlm.nih.gov/entrez/. Chose Structures
in the window Search and search for yor protein. Follow
the protein code via Download/Display File to PDB compression none. Download as described above. The are
also other ways to view proteins, i.e. Chime and Protein
Explorer, but in our hands only RasWin is flexible enough to give the freedom to side-by side use directly
Internet-connected, networked and free standing com-
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puter platforms necessary in a secondary school environment.
RasWin
To open the program
Enter the appropriate folder and start the RasWin program; normally Rw32b2a.exe. Pull out the window to
cover the whole screen. Note that you now have two
open programs; RasMol Command Line and RasMol
Version 2.6. We will start by taking a look at a small
protein and try out a few of basic commands in RasWin.
Commands
On top of the window you can see the menus File, Edit,
Display, Colours, Options, Export and Help.
File gives you the opportunity to Open or Close a data
file. An important feature in RasWin is the possibility of
selecting certain atoms for manipulation. Under Edit,
you can select all atoms in a data file with select all.
Display is more versatile. Here you find various options
to display the molecule. When a data file is first opened, it is shown in Wireframe, where all interatomic
bonds are shown. Try to Open the file Lysozyme, move
the molecule around by holding the left button down
and moving the pointer. Now use Display and shift to
Backbone to highlight the peptide bonds. Sticks is more
like Wireframe, but gives a better feeling of 3D-space.
Spacefill essentially shows the space occupied by the
electrons of the atoms and result in a close to true picture of the crowded interior of a protein. Ball & Sticks
depicts the protein much in the same fashion as the
ball-and-sticks models often used in school for smaller
molecules. Normally black is carbon, blue is nitrogen,
red is oxygen and yellow is sulphur, among the more
common atomic species. Ribbons show secondary
structure, i.e. alpha-helix and beta-structures. Strands
and Cartoons are variants of Ribbons, where especially
Cartoons enable easy tracing of amino acid sequences
in a complex protein.
Colours
Try the various settings of Colours, preferably in combination with the different settings in the Display-menu.
Monochrome gives black and white, CPK atomic standard colours, Shapely paints every type of amino acid
uniquely, Group assigns every separate part of the protein a separate colour, Chain gives each individual amino acid chains in a complex an individual colour, whereas Structure highlights alpha-helices and betastructures. Finally, Temperature indicates the degree of
mobility of individual atoms, depending on the availability of such information in the data file.
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The viewer
Choose Ribbons and Chain. Rotate the protein with left
button depressed, zoom by Shift+left button and move
sideways by pushing the right button.
Command Line
Click on a specific atom in the RasWin window and open
the RasMol Command Line to see the type and number
of atom targeted. In the command line-mode you can
select specified atoms, amino acids or chains for selective manipulation of presentation. Later, we will give
several examples of this function. Further details on the
function of Command Line can be found in the User
Manual.
Help
This manual reveals some of the options and functions
of RasWin. More information can be obtained under
Help User Manual. Feel free to read more and to experiment on your own.
End of RasWin session
File Close.
To study protein structures with RasWin
We will start by taking a closer look at a short polypeptide hormone; glucagon. It only has one type of secondary structure, but is useful to show some basic manoeuvres with RasWin.
Glucagon
Glucagon is formed by the alpha-cells in the pancreas and is
released when the level of
blood glucose is low. It consists
of 29 amino acids, and circulates in the blood where it
stimulates glucose release from
the liver.
Alpha-helix
Fig 1. Alpha-helix shown with ”ribbbons”(glucagon)
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File, Open, Glucagon. Try the
various settings of Display.
Spacefill shows the tightly
packed structure, without major pockets. Try also Colour, viz Shapely, which shows each of the 20 different
amino acids with their own distinct colour. It is evident
that glucagon forms an alpha-helix, and this can be
confirmed by Ribbons. Generally, the best way to obtain an overview of the structure of a protein is by Car-
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toons Structure.
Select individual amino acids
Ball&Sticks CPK. Open RasWin Command Line-window.
Write select trp followed by Enter and color red followed
by Enter. You have now selectively coloured tryptophane residues. Return to the RasWin-window. This
demonstrates the principle. After specific atoms are selected, all immediately following operations will affect
the selected atoms only.
Select a group of amino acids
Edit, Select all Ball&Sticks CPK Try to label all non-polar
amino acids. In the command-window, write select hydrophobic followed by Enter, return to the RasWinwindow Display, Ball & Sticks. You can now see the
non-polar amino acids distinct from the others. File Close.
Major features of proteins
Proteins with alpha- and beta-structures
Lysozyme, the enzyme in tear fluid degrading the cell
wall of certain bacteria, has both alpha- and betastructure. Open Lysozym. Cartoons Structure. Rotate
the protein try other Display and Colours settings. Another example is triose phosphate isomerase, the enzyme responsible for converting dihydroxyacetone
phosphate into glyceraldehyde 3-phosphate directly after the 6-carbon compound is cleaved into two 3carbon compounds in the glycolysis. Open Triose phosphate isomerase. Cartoons Structure. The central barrel
composed of beta-structures is typical of this enzyme.
A third and last example are immunoglobulines. Open
IgG Fab-fragment. Cartoons Structure. The protein is
composed of two distinct domains with a beta-structure
core. This domain type is used
in several other proteins of the
immune system and in growth
controlling processes.
Water-soluble proteins are only
polar on the surface
Fig 2. Myoglobin shown with ”spacefill”.
The heme group is green, polar amino
acids red, and hydrofobic amino acids are
shown in blue.
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Myoglobin is present in the
skeletal muscle as an oxygen
supply and is especially abundant in diving animals, like
spermaceti whales. Open Myoglobin. Cartoons Structure. Open Command Line and
write select polar Enter, followed by color red Enter. Go
to the RasWin-window. Spacefill . Go to the Command
Line, select hydrophobic Enter color blue Enter Space-
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fill select hem Enter color green Enter Spacefill and
under Mode choose Slab. Search through the molecule
slab by slab by moving the cursor with depressed left
button. See how the interior is dominated by non-polar
amino acids and how the periphery is mainly composed
of polar amino acids. You can also visualise the hydrophobic pocket where the relatively hydrophobic heme
group is bound. View the protein from outside by deselecting Slab.
What does an active site look like?
Trypsin is formed in the pancreas and degrades proteins in the food to smaller peptides, more suitable for
further degradation and uptake as free amino acids in
the blood stream. The active site of trypsin, as well as
of any other enzyme, has two distinct functions; to bind
the substrate in the active site, and to perform the catalysis. Trypsin has a preference to degrade peptides
and proteins adjacent to basic amino acids, that is arginine or lysine. This basic amino acid has a positive
charge on its side chain at normal pH. The catalytic
group of trypsin is the OH-group of a specific serine
amino acid. However, this serine requires the nucleophilic support from an aspartic acid and a histidine, and
these three important amino acids are collectively
called the catalytic triad.
Trypsin with benzamidine
Open Trypsin with benzamidine inhibitor. Ribbon. Label
the catalytic triad by select his57, asp102, ser195.
(Write them all in a row, with space after commas)
Spacefill. Demonstrate the bound inhibitor benzamidine
by select hetero and not hoh, Spacefill, Groups. The
binding site of the basic part of the lysine or arginine of
the substrate is exactly in the position of the bluecoloured inhibitor. The inhibitor functions by blocking
the binding of substrate to that particular part of the
active site.
Trypsin with protein inhibitor
Fig 3. Trypsin with inhibitor on the active
site.
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Open Trypsin with pancreatic
inhibitor: Ribbon, Chain, select
asp102, his57, ser195 Enter.
Spacefill. Again, the catalytic
triad is visible. The inhibitor
fills the whole active site, and
excludes all foreign substrates
from entering. The formation of
trypsin simultaneously, and in
complex with its inhibtor allows
the pancreas to escape auto
digestion. select lys15i, asp189e Enter Spacefill, Chain.
This demonstrates the lysine residue on the inhibitor,
resembling the basic residue of substrates, bound to
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Vol 2 ⏐ No 2
the negatively charged aspartic acid residue in the bottom of the active site in the trypsin molecule
Summary
You have now studied three main characteristics of proteins; secondary and tertiary structure, polarity and active site. You have only scratched the surface of what
there is to know about protein structure. This field of
knowledge is vast, and research is expanding towards
new horizons. If you wish to continue exploring the field, get hold of a standard biochemistry textbook, or
even better find an interested teacher or researcher.
Good luck!
The original version of this material was developed for chemistry in
higher classes of the secondary school. It is aimed to facilitate the
display of basic features of proteins in chemistry and some important
concepts in protein chemistry. This version is limited to fit into a single 60 min lesson. I am grateful to Karin Sandelius Hellström and
Paul Strömqvist as well as teachers and students at Rudbecksskolan,
who have contributed with critical comments and general support.
Any errors that remain are mine. I welcome all comments on the material and suggestions for improvements by email;
[email protected]
Anders Hansson
[email protected]
Illustrations from the program, RasWin
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