PROTEIN STRUCTURE TUTORIAL

The following tutorial was contributed by Andrew Coulson, Univ
Edinburgh UK (a.coulson@ed.ac.uk).  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).  It ends with an open-ended exploration of a DNA-binding
protein.

Practical 6

Protein Modelling

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 completing and writing
up the exercise, you should be able to

1. 	Use a molecular-graphics system to analyse the architecture of proteins.
2. 	Recognize the main elements of secondary structure within a protein fold.
3. 	Explain how secondary structural patterns have been used to
classify protein  structures.
4. 	Describe the main structural classes of globular proteins.

The main part of the practical is a 'guided tour' of seven proteins,
each typical of its class.  In the course of this tour you should also
become familiar with the features of the  graphics program 'RasMol'
which are useful for highlighting features of the structures.   The
final part of the practical is an open-ended exercise in which you
make use of these  tools to describe the structure and mode of binding
of a DNA-binding protein to its target  sequence.  

The microcomputer lab where this practical is being held is generally
available for  individual use, and  you are also encouraged to make
use of the lab facilities in your own  time to look at these
molecules, or any others in which you may be interested.  Please ask 
a demonstrator if you want to work on any other protein, and we will
get the co-ordinates  of any available solved structure.

You should take notes of what you observe, with sketches and diagrams,
if you find these  useful; it is also possible to get monochrome
prints from the graphics screen (ask a  demonstrator). At various
points in these notes there are specific questions for you to  answer.
If you choose to present your write-up of this practical for
assessment, there  should be a brief clear account of what you have
seen, and answers to the specific  questions.  You will find
discussion of some aspects of the structures of all these proteins  in
Branden and Tooze, Introduction to Protein Structure.


2. Molecular Graphics Program, RasMol.

RasMol takes as input a file containing the 3-dimensional coordinates
of the atoms in a  molecular structure; the program draws a
representation of various types of model.

The most realistic model represents each atom as a solid sphere
(hydrogen atoms are  usually omitted, and the size of non-hydrogen
atoms is therefore exaggerated a little so  that the space-filling
properties of groups and molecules are roughly correct). 
	
'Ball-and-stick' models allow you to see through the structure; atoms
are represented as  small spheres, and the bonds joining them as rods.
In the 'wireframe' model the atoms are  shrunk to the points where the
bonds (represented as wires) meet.  In the 'backbone'  model all the
side-chains are removed, and the folding of the polypeptide or nucleic
acid  chain is shown by straight rods joining the positions of the
centres of the a-carbon  or  phosphate atoms. The simplest model is a
smooth ribbon drawn through the peptide units;  this may be either
solid ('Ribbons') or made of parallel thin wires ('Strands')

The model can be rotated about the x, y and z axes interactively so
that all parts of the  molecule can be studied. The molecule can be
made smaller or larger  and in addition it is  possible to expand the
viewing window up to the full size of the screen  -  but the larger 
the picture, and the more elaborate the model, the longer it takes the
computer to  calculate the appearance of the drawing. A good technique
is to adjust the point of view  and other features of the drawing
using a small window and a backbone model, so that,  for example,
changes in the viewing position are made quickly, and then to make the 
model more complicated and the window larger so that the fine detail
can be seen.

Several colouring schemes are available; the four most useful are
'CPK' (carbon atoms  pale grey; oxygen red, nitrogen blue and sulphur
yellow), 'group' colouring (the chain is  coloured with the colour of
the rainbow, from blue at the N-terminus to red at the C- terminus;
this is useful for following the fold from one end of the chain to the
other), and  'shapely' and 'amino' colours.  In the former scheme, the
backbone is pale grey, and the  side-chain atoms are all given a
colour which depends on the size and the polarity of the  side-chain. 
Oxygen-containing side-chains (acids, amides,  and the
hydroxy-aminoacids  Ser and Thr) are various shades of red, and basic
side chains (Arg, Lys, His) blue.  Hydrophobic aminoacids are mostly
grey, but Ile is dark green and Val a pale magenta.  The
sulphur-containing amino-acids (Cys, Met) have muddy yellow colours,
Trp is  yellow and Gly, white.  The full list is:
	
	A  (ala) pale green	   	M (met) plale brown	
	C  (cys) sandy yellow	N  (asn) salmon
	D  (asp) dark magenta	P  (pro) grey
	E  (glu) red			Q  (gln) flesh pink
	F  (phe) grey			R  (arg) navy blue
	G  (gly) white			S  (ser) tomato
	H  (his) slate blue		T  (thr) orange red
	I  (ile) dark green		V  (val) pale magenta
	K  (lys) royal blue		W (trp) yellow
	L  (leu) grey			Y  (tyr) clay grey	

The 'amino' scheme is similar but simpler: E and D are red; K and R
blue; C and M  yellow; H unfaded blue jeans; Q and N a bright
green/blue; S and T orange; P flesh; I,L  and V dark green; W dark
purple; Y and F a lighter purple; G and A are off-white and  light
gray respectively.

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. You will be provided with a copy of the user
manual for the program, which  describes how all these features can be
used, and all this information is also available  using the on-screen
'Help' facility; however, it is often quicker to learn the techniques
by  demonstration than by reading about them, and the demonstrators
will try and move  round quickly to do so!


3. Use of RasMol: 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.

Start RasMol by clicking with the right button on the background; drag
down and to the  right to highlight 'Applications', 'Molecular
graphics' and finally 'rasmol'.  In the RasMol  window, click on
'Load'; in response to the filename prompt in the interaction window, 
type
	5RSA.pdb  <enter> 	
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 <enter>
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?

You can cut sections through the molecule by giving commands such as 
	slab 70
in the interactive window; this will cut away the front
30% (= 100% - 70%) of the  molecule.  Give the commands

	slab 20
	slab 30
	slab 40
	slab 50
	.....  etc  .....

to cut a series of sections from front to back. As you work you way
forward, sulphur- containing residues (yellow) appear. Some of these
are methionines, but there are also 8  buried cysteins, linked by 4
disulphide bridges. Identify these cysteines. Finally, give the 
command
	slab off
to turn off this feature.

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.


4. Helix proteins: b562 and myoglobin

Next we look at two alpha proteins, in which the main elements of
secondary structure present are a-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 a-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 N- terminus.  Finally, give the command:

	zap


5. Beta proteins:  crystallin and retinol-binding protein

The next two proteins have anti-parallel b-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 b-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 b-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 b-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 b-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, with diagrams (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 b-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?


6. Alpha/beta proteins: TIM and flavodoxin

The final pair of proteins are a/b 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 a-helices
and strands of b-sheet.   These elements are in strict sequence along
the chain, and should make a clockwise turn  from N-erminus 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 b-a-b units are assembled into the complete
barrel.

Finally, display flavodoxin in the file 4FXN.pdb.  Describe the
overall structure; display  a b-a-b 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 b-a-b 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 b-sheet. Which atoms
and groups are involved in binding the  coenzyme? 


7. Open-ended exercise on DNA binding protein.

The file  2CRO.pdb  contains the structure of a DNA-binding protein,
and 3CRO.pdb  a  proposed model of the way it binds to DNA. Use the
facilities of RasMol to describe the  folded structure of the protein,
and the way it binds to DNA. 

This protein binds to a specific sequence of bases. How might it
recognise where to bind?  The interaction between protein and DNA is
best seen by displaying the DNA as  wireframe, one subunit of the the
protein as backbone, and the other as spheres. Ask a  demonstrator if
you need help with this.



===== end of tutorial =======