| ## Getting started - Protein unfolding Main Table of Contents | VERSION 4.0 Sun 18 Jan 2009

### Protein unfolding

In this exercise we will study a protein unfolding simulation that was done before. The protein is the C-terminal fragment of the L7/L12 ribosomal protein (see below). It consists of 68 residues, and is known to be quite stable (in simulations). It is dissolved in a box filled with 3777 water molecules a structural Sulfate ion and four Sodium ions. A simulation was performed for 10 ns at 400 K. The trajectory and other relevant files can be found in `~david/ctf`.

Native structure | 200 ps
500 ps | 1 ns
4 ns | 10 ns

### Analysis

1. Start by making a new working directory, and then move there.

   | `cd ~/tutor`
   | `mkdir unfold`
   | `cd unfold`

2. View the trajectory on your own X-screen (program ngmx).

   | `ngmx -s ~david/ctf/unfold.tpr -f ~david/ctf/unfold.xtc` |

   Hint 1: In the filter it may be advantageous to select Mainchain rather than Protein.
   Hint 2: Go to the display menu and select options. Then set skip frames to 9 before you start the animation. What happens to the protein?

3. The Root Mean Square Deviation (RMSD) with respect to the crystal structure (program g_rms) is a measure of how well the crystal (starting) structure is maintained in the simulation.

   | `g_rms -s ~david/ctf/unfold -f ~david/ctf/unfold -o rmsd` |

   Select the 1 for the number of groups, and select C-alpha (group 3) for fitting and for computing the RMSD. View the output graph with xmgrace.

   | `xmgrace rmsd.xvg` |

   Does the RMSD converge within the simulation? If not, what does this indicate?

4. The Radius of Gyration (Rg, program g_gyrate)) is a measure of the size of the protein.

   | `g_gyrate -p -s ~david/ctf/unfold -f ~david/ctf/unfold -o gyrate` |

   Select protein when asked. View the graph with xmgrace:

   | `xmgrace -nxy gyrate.xvg` |

   Does the radius of gyration change during the simulation? The x, y, and z components indicate the overall shape of the molecule (like the axes of an ellipsoid). i.e. if they are all equal, the molecule has spherical shape, if one is much long than the other two, the molecule is elongated. Based on this graph and the animation does the protein change shape?

5. The Ramachandran Plot shows whether the backbone torsion angles (φ/ψ) of your peptide are within the allowed region. (program g_rama). We will compare the start structure and the final structure by running the program twice.

   | `g_rama -s ~david/ctf/unfold -f ~david/ctf/unfold -o rama-start -e 1` |
   | `g_rama -s ~david/ctf/unfold -f ~david/ctf/unfold -o rama-end -b 9999` |

   View the graphs with xmgrace:

   | `xmgrace rama-start.xvg rama-end.xvg -legend load` |

   In black we have the backbone angles from the starting structure, in red those from the final structure. Hint 3: click on the red graph, and a dialog box will plop up. Select linetype none for the second graph, and select a circle as a symbol. Are all the angles in the allowed region? What kind of structures do the angles indicate in the folded respectively unfolded conformation?

6. Now we will analyse the number of hydrogen bonds the protein makes. First with itself, then with the solvent.

   | `g_hbond -s ~david/ctf/unfold -f ~david/ctf/unfold -num hbnum-pp` |

   Select protein as the first group and second group. Then redo the analysis for protein with solvent (change the output file name to hbnum-ps, and select first the protein, and then solvent).
   View the output file:

   | `xmgrace hbnum-pp.xvg hbnum-ps.xvg` |

   Does the number of hydrogen bonds change for either of these?

7. Here we will analyse the solvent accessible surface area of the protein. We will be looking at both hydrophobic surface area and hydrophilic surface area.

   | `g_sas -s ~david/ctf/unfold -f ~david/ctf/unfold -n ~david/ctf/index -skip 25` |

   (Select protein again). View the output file:

   | `xmgrace -nxy area.xvg` |

   How do the two components of the solvent accessible surface area change? How does the total change?
8. Secondary Structure analysis (program my_dssp). This analysis uses the dssp (dictionary of secondary structure in proteins, Kabsch & Sander, 1983) software.

   | `my_dssp -s ~david/ctf/unfold -f ~david/ctf/unfold -dt 50` |

   Select protein when asked to select a group. You can postprecess the output file with:

   | `xpm2ps -f ss.xpm -o ss.eps` |

   This will give you a postscript file which you can either print or view with xpsview.

   | `xpsview ss.eps` |

   What happens to the Alpha helix (in blue)? What happens to the Beta sheets? Which secondary structure element is more stable?

9. Give a summary of what happens during the unfolding process. What happens first to the structure? How do the structure and shape of the protein develop? Try to formulate relevant conclusions for the protein folding problem based on this simulation.