Download Analysing a protein structure

Analysing a protein structure
Introduction
Primary structure
In biochemistry, the Primary structure of a protein is the exact specification of its atomic
composition and the chemical bonds connecting those atoms (including stereochemistry). Typically,
the primary structure is equivalent to specifying the sequence of its monomeric subunits, e.g., the
amino acid sequence.
Primary structure is sometimes mistakenly termed primary sequence, but there is no such term, as
well as no parallel concept of secondary or tertiary sequence. By convention, the primary structure
of a protein is reported starting from the amino-terminal (N) end to the carboxyl-terminal (C) end.
Secondary structure
In biochemistry and structural biology, secondary structure is the general three-dimensional form
of local segments of proteins. It does not, however, describe specific atomic positions in threedimensional space, which are considered to be tertiary structure. Secondary structure is formally
defined by the hydrogen bonds of the biopolymer, as observed in an atomic-resolution structure. In
proteins, the secondary structure is defined by patterns of hydrogen bonds between backbone amide
and carboxyl groups (sidechain-mainchain and sidechain-sidechain hydrogen bonds are irrelevant).
Tertiary structure
In biochemistry and molecular biology, the tertiary structure of a protein or any other
macromolecule is its three-dimensional structure, as defined by the atomic coordinates. Proteins are
capable of diverse functions ranging from molecular recognition to catalysis. Such functions require
a precise three-dimensional tertiary structure. While such structures are diverse and seemingly
complex, they are composed of recurring, easily recognizable tertiary structure motifs that serve as
molecular building blocks. Tertiary structure is considered to be largely determined by the
biomolecule primary structure, or the sequence of amino acids of which it is composed.
Quaternary structure
In biochemistry, quaternary structure is the arrangement of multiple folded protein or coiling
protein molecules in a multi-subunit complex.
Exploring the primary structure
In the Welcome window choose Protein structure. The beta-hydroxysteroid dehydrogenase will
appear.
You can see the primary structure in Discovery Studio Visualizer by opening the Sequence Window
(Ctrl+Q)..
Exploring the secondary structure
Make visible the secondary structure on the Sequence window: choose Sequence | Secondary
Structure | Visibility, then select only PDB and press OK. The secondary structure feature will be
shown. (e. g. alpha-helices in red & orange stripes)
The alpha-helices are stabilized by H-bonds between the peptidic hydrogen of one residue and the
carbonylic oxygen of the 4th-position before residue.
Now try to visualize this H-bonds.
Firstly select a sequence in an alpha helix conformation (for example select the sequence 98-133)
Activate the graphic workspace windows clicking on the tab (where there is the name of the protein) so
the selection remains. In the Display Style window (Ctrl+ D) select Atom | Line, then deselect the
sequence by clicking in a empty region of the workspace and in the Display Style window select Protein
Off. You should have on the screen the portion selected before in a wire representation.
If also ligands appear you can select them by clicking twice on them and pressing the Canc key.
At this point select through the mouse the visible portion of the protein and label the residues: choose
Structure | Labels | Add | Object = AminoAcid, Attribute = 3-Letter & ID#. Then add the polar
Hydrogens and visualize the intramolecular H-bonds. (Structure | Monitor | HBonds)
Save the so obtained image (choose File | Save As, give a name and choose Image File). You can save
in the same manner every active screen.
You can see the H-bond network with 1-5 interaction: for example the carbonylic oxygen of Ser109 is
H-bonded to the peptidic hydrogen of Val 113.
The β sheet (also β-pleated sheet) is the second form of regular secondary structure in proteins, only
somewhat less common than the alpha helix. Beta sheets consist of beta strands connected laterally by at
least two or three backbone hydrogen bonds, forming a generally twisted, pleated sheet. A beta strand
(also β strand) is a stretch of polypeptide chain typically 3 to 10 amino acids long with backbone in an
almost fully extended conformation.
Restore the initial visualization of the beta hydroxysteroid dehydrogenase (unselect all by clicking in an
empty zone of the workspace, then on the Display Windows select Atom=None, Protein=Solid Ribbon,
Label=Off). You can visualize the sheet as light blue portion of the ribbon on the graphic window and
as light blue arrows in the Sequence Window.
Clicking on a sheet in the graphic window you can see on the Sequence which is the related portion.
Select on the Sequence all the residues of this sheet portion the on the Display Style do Atom=Line.
Repeat this action on the three sheet portion. Now unselect (clicking in the workspace) all and hide the
ribbon (Display style, Protein = Off). You should see only the atoms of the three selected sheet portions
Then Visualize the H-bonds: choose Structure | Monitor | HBonds.
You can monitor the H-bond distances (firstly, through the mouse select all visible atoms, then Structure
| Labels | Add Object=Hbond, Attribute=Distance). Save the image.
Exploring the tertiary structure
The tertiary structure of a protein is determined by interactions among far residues. You can find Hbonds, hydrophobic interaction, electrostatic interaction and, in certain cases, also S-S covalent disulfide
bonds between two cysteines.
In the Display Style window choose Atom=None, Protein=Tube, Color by=Hydrophobicity. You can
see that hydrophobic residues (Brown) stand in front of hydrophobic residues and that polar residues
(Blue) stand in front of polar residues and these interactions are in some extent responsible for the
folding of the protein. Remember that the hydrophobic/hydrophilic properties of residues are due to
their side chains.
The H-bonds determining the tertiary structure can be searched. For this purpose use the tube
representation of the protein while all atoms are hided and add polar hydrogens (Chemistry | Hydrogens
| Add polar) . Then Structure | Monitor | HBonds. You see nothing because the H-bonds are visualized
only if the atoms are visible. Therefore select through the mouse a portion of the protein in which two
portion are close and probably interacting, then display the atom as line. At this point you can see Hbonds if there are.
In this protein there are not disulfide bonds. If they are you can find them in the Hierarchical Windows
by expanding protein groups. (View | Hierarchy)
Ramachandran plot
The geometry of a protein is mainly determined by the angles Phi, Psi and Omega, but the omega angle
is almost stable due to the conjugation of the peptide bond, so only phi and psi angles can vary and
determine the whole protein secondary structure.
Not all couple of psi and phi values are equally allowed due to the steric hindrance of the site chain.
The so called Ramachandran plot takes into consideration in a 2-D plot all the possible values of the phi
and psi angles showing the more or less allowed region of the conformational space.
Choose Chart | Ramachandran plot. Regions surrounded by the blue line are very favorable, regions out
of the violet line are unfavorable, regions between blue and violet lines are generously favorable. The
circles represent all the residues (triangles are glycine, squares are prolines).
If a residue is selected in the graphical window it is also selected in the Ramachandran plot and
viceversa-
You can note that the conformations of the low left part of the plot mainly correspond to the helix
secondary structure while those of the high left part mainly correspond to beta-sheet secondary
structure. The only residues in unfavorable regions are glycines which are the only residues without side
chain.
Exercise
Repeat the tutorial for another protein which you can download from Protein Data Bank with the code
2X4U. File | Open URL… Code=2x4u. You need to be connected to Internet!.
Simplify the structure only considering the first A chain: delete all other chains.
Then write a brief report about your analysis of this protein and save it in a file named as your
Surname+First letter of your name+numer 1; i.e. my file would be named MartinelliA1.
The allowed file types are only .doc, .rtf, .pdf.
This report should contain some Figures with captions (see as an example the file MartinelliA1.pdf
concerning beta-hydroxysteroid dehydrogenase).
Caution! This enzyme contains disulfide bonds and you should describe also them. Remember that you
can visualize disulphide bonds by Hierarchical windows in Protein Groups.