Download Lab #5 - bu people

EK-130
Lab 5: Protein structure
A. Program
Install RasMol + manual from
http://www.umass.edu/microbio/rasmol/getras.htm
(They may be already installed):
B. Tutorials
Look at the secondary structure tutorial at
http://www.umass.edu/molvis/freichsman/SecStrTut/Helix/menu.html
Another good tutorial on protein structure:
http://webhost.bridgew.edu/fgorga/proteins/default.htm
C. Tests
Complete the self assessment quizzes on secondary and tertiary structure of proteins:
http://pps9900.cryst.bbk.ac.uk/self-assess/sess1/pps00frm.html
http://pps01.cryst.bbk.ac.uk/self-assess/pps01quiz2.html
If you need help, read the protein structure tutorials
http://www.cryst.bbk.ac.uk/PPS2/course/index.html
http://bmbiris.bmb.uga.edu/wampler/tutorial/prot0.html
D. Lab and HW 5: Protein Analysis
The following tutorial is based on the exercise developed by Andrew Coulson. It teaches
the student to recognize the main elements of protein secondary structure, and the main
structural classes of globular proteins. The tour begins with an examination of RNAse,
continues with two alpha proteins (cytochrome B562 and myoglobin), two beta proteins
(crystallin and retinol-binding protein), and two alpha/beta proteins (triosephosphate
isomerase and flavodoxin).
1. Objectives
The object of this exercise is to allow you to become familiar with the main architectural
features of several classes of globular protein, by studying computer-generated
interactive drawings of
models of the proteins. We hope that this will make the descriptions of protein
structures in course lectures and in textbooks such as Branden and Tooze more
comprehensible. After the exercise, you should be able to
1.
2.
3.
Use a molecular-graphics system to analyse the architecture of proteins.
Recognize the main elements of secondary structure within a protein fold.
Explain how secondary structural patterns have been used to
classify protein structures.
4.
Describe the main structural classes of globular proteins.
2. Download protein structures
Go to the website:
http://www.rcsb.org/pdb/
Find and download the following protein structure files
5RSA
256B
1MBN
4GCR
1RBP
1TIM
4FXN
When naming the files, use extension pdb, i.e., 5RSA.pdb, 256B.pdb, etc.
3. Start the Molecular Graphics Program RasMol
Several colouring schemes are available; the four most useful are 'CPK' (carbon atoms
pale grey; oxygen red, nitrogen blue and sulphur yellow). The properties of the drawing
are changed either by picking items from menus with the mouse, or by typing commands
into a second window. Clicking with the mouse on any atom will cause the atom to be
identified in the second window. Drawings can be made using selected parts of the
molecule only; and it is often useful, for example, to draw some atoms or groups with
the space-filling representation and others as wireframe or backbone. H-bonds
(involving the backbone of proteins or the bases of nucleic acids) and disulphide bonds
can also be drawn, to help show secondary structure. The program knows how to
identify secondary structure, and so these too can be isolated and drawn differently.
4. Analysis of Ribonuclease
The first molecule to look at is the enzyme ribonuclease, which catalyses the hydrolysis
of RNA. We shall see how to use some of the features of RasMol, and in particular how
simplifying the structure makes the architecture of the fold more comprehensible.
In the RasMol window, click on 'File'; and ‘Open’ 5RSA.pdb. Click on 'Display' and
then 'Spacefill'. Open the 'Options' menu, and highlight 'Hydrogens'; the effect should be
to remove the hydrogen atoms from the model. Note that some water molecules are
shown; these are molecules which are fixed in the crystal stucture and therefore appear
in the image. Remove them by giving the commands:
select DOD
spacefill off
select amino
(Can you suggest why you select DOD, rather than HOH?)
Note the prominent cleft in the surface, and the bound phosphate (orange and red) ion
near the middle of the picture. Just to the right you should see the 5-membered ring of a
histidine sidechain.
Click on one of these atoms to confirm that this is His-119. This is one of the two His's
in the enzyme active site. The other is concealed by the phosphate ion; remove the latter:
select [PO4]
spacefill off
select amino
Now find the other histidine, His-12. To what extent is each of these side-chains buried,
or exposed to solvent?
Change the view of the molecule by giving the following three commands
in the interaction window (note that the x-axis is across the viewing window, the y-axis
vertical and the z-axis perpendicular to the screen). Keep your eye on His 119 during
these operations, and if you lose it, go back by changing the sign of the angle of rotation
until you find it again...
rotate z 110
rotate x -15
rotate z 15
Note that the cleft is now vertical, and that sulphur atoms are visible to the right.
Click 'Options' and then 'Shadows' - this gives a better impression of the 3-D structure,
but slows down the drawing. What shape is the cleft? Click 'Options' then 'Shadows' to
turn off this feature.
Give the command
Colour atom amino
in the interaction window. Are there any other potentially chemically active side chains
near the active site? List some of the residues which line the cleft. Looking overall at the
surface of the molecule, do you expect ribonuclease to be an acidic (pI < 7) or basic (pI >
7) molecule?
Click 'Colours', then 'Group' to see the run of the polypeptide chain from N-terminus
(blue) to C-terminus (green).
Now simplify the representation by:
Click on 'Display', then 'Ball-and-Stick'
Click on 'Display' then 'Wireframe'
Click on 'Display' then 'Backbone'
Click on 'Display' then 'Ribbons'
Use the scroll-bars and mouse to rotate the molecule and list the secondary structure
elements in order down the chain. Draw the active site histidines again:
select (his12 or his119) and sidechain
wireframe 200
colour atoms red
Which elements of the secondary structure support His-12 and His-119? What supports
the rest of the active site cleft?
Finally, give the command
zap
to remove this molecule.
5. Helix proteins: b562 and myoglobin
Next we look at two alpha proteins, in which the main elements of
secondary structure present are -helices. The main purpose is to
study two ways in which helices are assembled to make a complete
protein, but we also consider other aspects of the proteins'
structure and function.
The first of these proteins is cytochrome b562, a haem-containing electron transport
protein. Load the file
256B.pdb.
Click on 'Display' then 'Backbone', Click on 'Colours', then 'Group'
Note that there are two molecules in this structure; isolate one of them with the
commands:
select *A
save temp.pdb
zap
load temp.pdb
Click on 'Display' then 'Backbone'. Click on 'Colours', then 'Group'
Use the scroll bars to rotate the molecule; notice that it consists of a bundle of 4 helices.
Sketch their general arrangement. Note that the inter-helix loops are shorter at one end of
the molecule than the other, and that this gives it an overall conical shape (draw spheres
to check). The haem is bound near the wider end of the cone. Notice how the helices are
packed against one another. Add H-bonds with the commands
colour hbonds type
hbonds 50
set hbonds backbone
Give the command
backbone off
hbonds off
in the interaction window. Give the command
select resno > 65
in the interaction window to cut off the N-terminal part.
Click 'Display' then 'Backbone'; note the N-to-C direction.
Click 'Colours' then 'Shapely'
Click 'Display' then 'Spacefill'
The command
set background white
may make things easier to see.
Give the command
select all
in the interaction window, then
backbone 100
to draw the rest of the backbone. Rotate the molecule to see which residues are in contact
with solvent, and which form a hydrophobic patch between the helices; identify the
residues in the interior line of hydrophobics.
Go back to the backbone drawing:
Click 'Display' then 'Backbone'
Click 'Colours' then 'Group'
Note and sketch the angles between the axes of the pairs of helices. Give the command
zap.
The second -helix protein is myoglobin, the haem-containing oxygen binding protein of
peripheral tissue. The structure is very similar to those of the subunits of haemoglobin.
Re-start RasMol with the file
1MBN.pdb
Note from the wireframe drawing that the haem group is visible in the bottom centre of
the drawing.
Rotate the molecule so that the haem is edgewise on and just above the middle. Draw the
molecule in amino colours and spacefilling.
Click 'Options' then 'Het Atoms'. Note that the haem-binding pocket is now visible.
Click 'Options' then 'Shadows'. Identify the residues in contact with the haem.
Draw the backbone in 'group' colours. Count the helices; which helices support the
haem-binding pocket? Turn the backbone off and give the commands
select resno>57 and resno<79
in the interaction window to isolate part of the structure. Display it as backbone and then
as spheres, in shapely colours. Give the commands:
select all
backbone on
in the interaction window. Note the complex 'lumpy' surface of the helices and the
hydrophobic residues at helix crossing points. Identify these, and sketch the crossing
angles of the helices in contact. Go back to a complete backbone drawing and sketch the
molecule as a 'bent sausage'. Identify the helices by letters A,B,C etc from the Nterminus. Finally, give the command:
zap
6. Beta proteins: crystallin and retinol-binding protein
The next two proteins have anti-parallel -sheets as their main secondary structural
elements. The first is an eye-lens protein called g-crystallin.
Start RasMol with the file
4GCR.pdb
Display the molecule as a group-coloured backbone and rotate it to see that it is formed
from two distinct domains. Isolate the second half of the chain by giving the following
commands in the interaction window:
wireframe off
select resno>82
backbone 100
select selected and sheet
ribbons on
colour group
centre val126.N
Rotate the molecule to see the secondary structure elements are linked together; a good
view is looking up from the bottom of the 'bell'. Describe the fold; sketch a diagram
showing the orientation and order of the -strands. When writing up, compare this
diagram with that on p.68 of Branden and Tooze; confirm that the domain contains two
Greek key motifs.
'Buried' defines a class of aminoacid - those that are usually buried, out of contact with
the solvent. Give the commands:
select buried
resno>82
Click 'Display' then 'Spacefill'. Now give the commands:
select resno>82
backbone 100
Note that the -sheet forms a hydrophobic sandwich. Finally display the second half of
the molecule and confirm that the number and order of the strands (i.e. the topology) of
this domain is the same as the first.
The second -sheet protein is retinol-binding protein, whose function is to bind vitamin
A. Display the structure, in file
1RBP.pdb
The structure can be described as a back-and-forth -sheet, rolled into a sandwich. The
following commands will display the fold:
wireframe off
set background [230,230,230]
select sheet
ribbons on
colour group
set bondmode and
hbonds 70
colour hbonds type
set hbonds backbone
Describe the structure of the fold (Hint: start by looking at residues 22 to 106, and then
add the rest of the chain in segments).Describe how the barrel is made up; what shape is
its cross-section? Draw the bound retinol molecule by giving the commands:
select RTL
Click 'Colours' then 'Shapely'
Click 'Display' then 'Spacefill'
Where does retinol bind with respect to the -sheet? Rotate the molecule so you have a
broadside view of the retinol. Give the following commands to see how retinol is bound:
select all
Click 'Display' then 'Wireframe'
Click 'Colours' then 'Shapely'
Click 'Display' then 'Spacefill'
slab 55 [you may need a slightly different value]
Click 'Options' then 'Hetero'
Give the following commands:
select amino
Click 'Colours' then 'Shapely'
Click 'Display' then 'Spacefill'
slab 55 [you may need a slightly different value]
select RTL
spacefill on
Which parts of the protein are in contact with retinol? How do you think the retinol gets
into its hole?
7. Alpha/beta proteins: TIM and flavodoxin
The final pair of proteins are / proteins, in which the secondary structural elements are
alternating helices and strands of beta-sheet. Display triosephosphate isomerase, in the
file 1TIM.pdb. Display the backbone of one subunit ('select *A') in group colours. Rotate
the molecule to see that it is a drum of alternating -helices and strands of -sheet.
These elements are in strict sequence along the chain, and should make a clockwise turn
from N-terminus to C-terminus. Display the molecule as spheres to see the overall shape.
Go back to a backbone drawing, and then
select Ile or Val or Leu
and display spheres; observe that the branched-chain hydrophobic residues provide the
packing between the helixes and the sheet. Repeat with
select hydrophobic
to see how the rest of the core of the molecule is made up. Go back to a backbone
drawing and rotate through 90 about the y-axis. From the side view of the barrel, note
the direction of the strands and the helices with respect to the axis of the barrel. Note that
the loops are smaller and tighter at the right hand end of the molecule (the end you were
looking at), giving an overall shape of a truncated cone.
The active site is at the other end of the molecule, built from the longer loops (which are
presumably not so essential to maintaining the overall structure of the fold). Rotate
through another 90 , and display the molecule as spheres, with shadows and with shapely
colours. The active site is the central depression, and the catalytically active residues are
Glu-165, His-95 and Lys-13. To describe the underlying fold, display residues 1 to 44 as
backbone in group colours. Note the lengths of the a and b sections; as you go from N to
C terminus, do these elements describe a right-handed or a left-handed turn?
Display the rest of the backbone (thinner than this part). Sketch the way in which
successive  units are assembled into the complete barrel.
Finally, display flavodoxin in the file 4FXN.pdb. Describe the overall structure; display
a  unit (e.g. residues 80-120). What is the handedness (=left- or right-hand turn,
going from N to C) of this unit?
Display the complete molecule and note the angle made by successive strands of the
sheet to each other and to axes of the helixes. Draw a diagram to show how the --
units are connected together. What is the order of strands in the sheet? Note that the
longer loops are at the C-terminal ends of the beta strands.
select FMN
spacefill on
to see the binding site of the dinucleotide co-enzyme. Sketch the position of the binding
site with respect to the -sheet. Which atoms and groups are involved in binding the
coenzyme?
Additional questions:
1. Sketch a Ramachandran plot indicating regions of important secondary structure
motifs. Why might the distribution of main chain torsion angles observed in the protein
structures differ from those predicted by Ramachandran?
2. Which atoms on what residues form the backbone hydrogen-bond in an
(a) helical structure?
(b) beta-sheet?
(c) Type II beta turn?
(d) -turn?
e) 3-10 helix? .
3. Which residue causes an alpha-helix to kink or distort, and why?
4. (Partially) surface helices and surface beta sheets have characteristic hydrophobicity profiles
(i.e., hydrophobicity as a function of residue number). What is it:
a) For an alpha helix?
b) For a beta strand?