Mutations

Making a protein more stable

Before we do the more complicated things like homology modeling we will look at exchanges of a single residue at a time. Understanding which interactions are favourable for protein stability and which interactions are unfavourable is crucial for any (future) protein engineer, protein chemist, etc. In this step we will spend some time on making a protein more stable by means of point mutations.

Working with YASARA and the WHAT IF servers

In this course we will mainly work with the YASARA Twinset software. If, for what-ever reason, we cannot use the teaching room in the CMBI that has the powerful computers that can run the Twinset, we will have to improvise using an USB stick that holds the Twinset. Twinset is a version of YASARA that has all of WHAT IF built-in. (WHAT IF is rather hard-to-use software that can do thousands of things, like for example making mutations in proteins).

Figure 2. During this course we will make intensive use of the full, professional version of YASARA. At home you can use the freeware YASARA viewer. You can download this free viewer from the useful links, or you can request your own personal (commercial) version from www.yasara.com.

Figure 3. We will use the WHAT IF servers for a series of computations such as torsion angle determinations, homology modelling, structure validation, etc.

For this course I have made several special WHAT IF servers that can do the things one would normally do interactively.

Figure 4. The structure of crambin (PDBid=1crn). We will often use either crambin, or hypothase (a molecule I designed myself...) to work with.

So, first get familiar with the WHAT IF servers. Browse through them. To make sure that you look at several of them, answer question 1.

Question 1:

1. What are the coordinates of the C_α  of Phe-13 in 1crn?
2. What is the secondary structure of Gly-42 in 1crn?
3. What is meant with "contact distance" and how many residues make a contact with the C_γ2 of Thr-1 if a contact distance of 1.0 Ångström  is used?
4. Which cysteines are paired in 1crn?
5. How many residues make a tight (0.5 Ångström  cutoff) crystal packing contact in 1crn?
6. List the hydrogen bonds made by Thr-1 in 1crn.
7. List all torsion angles in 1crn, and explain the columns in the server output.

One mutation at a time

Before we do the more complicated thing like homology modeling we will look at mutations of a single residue at a time.

Understanding which interactions are favourable for protein stability and which interactions are unfavorable is crucial for any (future) protein engineer, protein chemist, etc. In this step we will spend some time on making a protein more stable by means of point mutations. Not that we actually want to do that in the real world, but these exercises will illustrate many important aspects of protein structure, function, and stability.

Protein stability

Proteins are stable if the equilibrium:

         Folded protein  <-->  Unfolded protein
                         or
                      F  <-->  U

is strongly shifted to the left. And to make a protein more stable, you 'only' have to shift that equilibrium a bit further to the left. That immediately implies that you can just as well destabilize the unfolded form, as stabilize the folded form. Both will get you where you want to be. Urea and similar denaturants , high or low temperature, organic solvents , and bad mutations generally will shift the equilibrium to the right.

We will not discuss here all aspects of protein stability. That would be an entire course in itself. However, the following rules of thumb hold rather often:

• To stabilize a protein you should know why it is unstable in the first place;
• Upon designing mutations, look for examples first, and only think yourself if nothing else works;
• Use residues that family members have already used at that spot;
• In line with the previous comment, look at a multiple sequence alignment ( MSA) for example a HSSP file (that can be found most easily using MRS) ;
• Helix capping is always a very good bet;
• Think about entropic stabilization (i.e. Gly ->  X or X ->  Pro) (Pro in loops is almost always a sure bet, if it fits);
• Add a Cys-Cys bridge (but make it geometrically perfect or you are in shit);
• Fill cavities (normally that does not bring much, but combined with entropic stabilization, i.e. Gly ->  Ala, it can be very effective;
• Add surface salt bridges;
• Get rid of buried water molecules (will often only work with Ala ->  Ser, but keep looking);
• Improve hydrogen bonds;
• Improve propensity of helices and propensity of strands;
• When you have choice between mutating a buried and a surface residue, take the surface one;

Read the supplemental material on stabilizing mutations for more explanations. This supplemental material is exam material (despite the gray background, sorry).

And now the real thing

Use everything available to you (don't forget that it is allowed to use your brains too at this stage) to think of stabilizing mutations. You can use the WHAT IF servers to produce coordinates with the mutation included, and YASARA for visual inspection. Make a list of your mutations, and write in a few words WHY you designed each mutation.

The multiple sequence alignment for crambin (1crn) is available in the Exercise files sectoion. At the bottom of that file I added, as a service to you, all torsion angles.

WHAT IF provides a nice option that can help you predict the side chain structure (rotamer) of a mutated residue. It works as follows: Hit the space bar (twice) and you get a text terminal window; the first time you do this in a YASARA session you should type WIF in this terminal window; you get the WHAT IF menu:

WIF - Switched to WHAT IF command syntax. Type 'YAS' to go back to YASARA. Click
outside the text window or type 'GO' to close the console.
HELP   INFO   SHELL  GENMEN END    $..  %..  !.. SCRIPT - MainMenu
DOLOG  NOLOG  GO   FULLSTOP LISTA  LISTR  HISTOR GETMOL -
GRAFIC GRATWO GRAEXT COLOUR PLOTIT USEGRA ITMADM LABEL  -
SOUP   3SSP   ACCESS ANACON BUILD  CHECK  CHIANG CLUFAM -
DGLOOP DIGIT  DOSELF DRUG   DSSP   ELECTR EXTRA  HBONDS -
HSSP   MAP    MODIFY NEURAL NMR    NOTES  OTHER  PROTON -
QUALTY REFINE SCAN3D SCNSTS SEARCH SELECT SEQ3D  SETPAR -
SETVDW SHOENT SPCIAL SUPPOS SYMTRY TABLES WALIGN        -
WATER  XRAY                                             -
ANATRA TRAMOV GRID                                      -
---------------------------------------------------------
WHAT IF>

Don't worry about this, just type:

1. %dgrota
2. The residue number
3. Give Return for the group number
4. Give the three letter code of the residue you want
5. Hit Return for the MOL-object
6. Hit Return for the Mol-item

And click in the graphics window to get rid of the text terminal. In the molecule you now see a big cloud of side chains in the molecule:

Figure 5. The rotamer distribution for phenylalanine at position 12 in crambin. An explanation is given in the linked material, and in these three short videos: , , and .

Question 2: Think of 5 very different reasons for making a mutation in a protein.

Question 3: Make a flow-chart with all points to think about when designing a mutation.