KE0026 Biochemistry Labs

Protein modelling tutorial

Litterature: Stryer chapter 3; Horton et al. chapters 3 and 4
Materials: Push-fit molecular models from Nicholson, Labquip, England

Introduction
The aim of this practical is to increase the understanding of three-dimensional structures of macromolecules. You will use a standardized system which consists of moulded plastic units indicating atoms or groups of atoms which can be rapidly assembled into high molecular weight molecules. Interatomic distances are automatically set by the depth of the socket in each unit. The scale is 1 cm = 1 Å (0.1 nm). An advantage with this system compared to computer graphics which you will also use during the course is that you get a much better 'hands on' feeling for atom connections, distances, sterical restraints etc.

Units for the polypeptide backbone
We will start this part of the excersise by identifying all the atoms in the units used to build the polypeptide backbone. This will help you to determine the direction of the polypeptide chain (N- or C-terminal) as well as the positions of side chains, etc.

The polypeptide backbone is made by connecting two different types of pieces. One represents the peptide bond and is constructed as a planar amide group with a socket (hole) in the carbon atom and an arm on the nitrogen for connection to the alpha carbon (Ca). It is not possible to rotate about the peptide bond which is between the carbon and the nitrogen. The second piece is a tetrahedral carbon that represents the Ca.

As you can see from the figure below the peptide bond unit contains parts from two residues - it has the carboxyl portion from one residue and the amino group from the following residue. There are two possible non-identical positions for the side chain (=R). Only one is correct for an L-amino acid. On page 43 in Stryer (53-55 in Horton) you will find a description of that.

Model building and questions
Each group will get three segments of poly-glycine sequences. Each segment has a common motif of secondary structure: parallel b-strands, antiparallel b-strands and an a-helix starting and ending with a loop. A few hydrogen bonds are marked with plastic tubing. We shall in detail study a- and b-structures and connect the segments to a protein structure with the topoplogy shown in the figure at the end of these instructions. In the figure, arrows represent b-strands and the cylinder an a-helix. Follow the instructions below and build your molecule at the same time.

Study the b-structure segments. Look carefully at the direction of the strands (N- and C-terminals are labeled in the figure) and the pattern of hydrogen bonds (Stryer p. 59-60; Horton p. 93).
What are the differences between parallel and anti-parallel b-strands?
Which atoms are involved in hydrogen bonding between the b-strands?
Connect the anti-parallel strands with a b-turn (Stryer p 60-61, fig. 3.42; Horton p. 93 fig. 4.18) to a b-hairpin. Which amino acids are most common in b-turns, and why?
Study the a-helix. Only a few hydrogen bonds have been indicated by plastic/rubber tubing. How many hydrogen bonds are formed from the first to the last marked bond in your helix ? What is the direction of the carbonyls -- are they pointing towards the N- or C-terminus?
What configuration of amino acids do you find in proteins (L- or D)?
Mark with a few methyl groups (grey tetrahedrons) the positions of the side chains.
Which amino acid has only a methyl group as a side chain and what properties does it have?
You are now going to connect the secondary structure elements to a continous peptide chain. For this exercise you need to look at the topology diagram below. Remember that the N- and C-terminals have been 'labelled' with blue and red pieces, respectively.
1. Connect the parallel b-strands with the a-helix to a compact entity.
2. Assemble the resulting pieces (now only two) to form one continous peptide chain in such a way that one of the parallel b-strands is anti-parallel to one of the other antiparallel strands. Connect with a b-turn and mark the H-bond in the turn with the tubing.
3. Use the topology diagram to check that your model is correct.
Now it is time to add some side chains.
Insert the following three sequences taking into account the properties of the side chains (size, hydrophobicity, charge etc.)
```
gly-gly-pro           ala-ser-leu             lys-gln-asp
```
When a new model of a protein has been built it is common to compute a so called Ramachandran plot. Why do we do that? (Stryer p. 56 fig. 3.28; Horton p 88-89).
What is a peptide plane?
Which bond in the plane is "stiff", and why?
Which are the only two torsion angles that can be rotated?
Read the approximate allowed values of phi and psi within a b-strand, a-helix and for the amino acid glycine.
Which different interactions are formed within a protein?
What is a b-twist (Stryer p. 60 fig 3.40)? Explain and make a b-twist on the b-pleated sheet of your protein model.

Lab index

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