Amino acids

Amino acids are at the heart of bioinformatics. Evolution of life is mainly governed by individual amino acid changes that slowly accumulate over the eons (this is not the whole story, of course, but surely a large part of it). If we want to align two sequences, we align amino acids. And your body's workhorses, the enzymes, consist of mainly amino acids, and if we want to modify a protein using protein engineering techniques, we exchange amino acids.

Therefore, a thorough understanding of the amino acids is needed. At the end of this course you will all know the twenty amino acids as if they are your friends. You will know their names, abbreviations, physico-chemical characteristics, and a whole lot more. At several moments we will relate things to thermodynamics and we will mention the "rule of 10":

The most important characteristic of amino acids is their hydrophobicity, but their size and charge are also important. The hydrophobicity is intimately connected to the role of water:

Figure 40. Many people have tried to make pictures that explain ALL amino acid properties in one shot. Obviously they all failed; just like this picture. Still, I hope that you come back to this picture at the end of this step, and explain how it got this shape. Be aware that this picture, originally designed by Willy Taylor more than 20 years ago, calls cysteine polar only because it can form (a very weak) hydrogen bond, and it doesn't know about intermediate size or intermediate hydrophobicity. I do not know why Histidine is in the hydrophobic circle, it certaily is very hydrophylic.

EU name: BIOBCK

Amino acid background material

The table below lists 5 items. You should now briefly look at this material, and study it either at your leisure at home, or when you need it while going through the questions later today or in the coming weeks. The five items are:

• Once more the amino acids. This is just one picture with the covalent structures of the amino acids shown. You can use this picture if you forgot to take the orange NBIC amino acid sheet.
• Physico-chemical properties. This is a pointer to a long, long list of tables with amino acid properties. In this physico-chemical properties section you can for now skip most parts, but you should look at the parts about solvent accessibility (1.5), chemical classification (3.1), hydrophobicity scales (3.2), and the genetic code (5.1). The other sections will be discussed later.
• The bedtime story about the amino acids explains the functional characteristics of the amino acids. This part overlaps with the seminar, but here you get it explained by somebody else, and sometimes that helps clarify things.
• Information for the real fanatics The information for the real fanatics forms nice reading, but does not contain material required to do well in the examination.
• The infamous amino acid test is listed so that you can print it when needed/desired.

EU name: AATYP

Working with amino acid structures

These small amino acid pictures will start rotating if you click them.

1. Alanine
2. Cysteine
3. Aspartic acid
4. Glutamic acid
5. Phenylalanine
6. Glycine
7. Histidine
8. Isoleucine
9. Lysine
10. Leucine
11. Methionine
12. Asparagine
13. Proline
14. Glutamine
15. Arginine
16. Serine
17. Threonine
18. Valine
19. Tryptophan
20. Tyrosine

Question 33:

Figure 41. Which residue is this, and what do you know about its special characteristics?

Question 34:

Figure 42. Which residue is this, and what do you know about its special characteristics?

Question 35:

Figure 43. Which residue is this, and what do you know about its special characteristics?

Question 36:

Figure 44. Which residue is this, and what do you know about its special characteristics?

Question 37:

Figure 45. Which residue is this, and what do you know about its special characteristics?

Question 38:

Figure 46. Which residue is this, and what do you know about its special characteristics?

Question 39:

Figure 47. Which residue is this, and what do you know about its special characteristics?

Question 40:

Figure 48. Which residue is this, and what do you know about its special characteristics?

Question 41:

Figure 49. Which residue is this, and what do you know about its special characteristics?

Question 42:

Figure 50. Which residue is this, and what do you know about its special characteristics?

At this moment you should know your amino acids. before continuing the course, do the amino acid quiz.

The Quiz

Question 43: Below you see all but the last quiz-question repeated in almost the same words as used in the quiz. You don′t have to write down the answers because the quiz-system tells you whether your answers were correct or wrong.

1. Which is the largest standard amino acid?
   
2. Which is the most acidic standard amino acid?
   
3. Which is the smallest standard amino acid?
   
4. Which standard amino acid has no chiral center?
   
5. Which standard amino acid contains an unusual carbon nitrogen bond making it an imino acid?
   
6. Which standard amino acid is the best buffer under physiological conditions where pH = 7.0?
   
7. Which standard amino acid has the longest aliphatic sidechain branched at the beta carbon?
   
8. Which of the following amino acids is not aromatic (PWYF)?
9. Which standard amino acid can covalently dimerize spontaneously?
   
10. What is the C-terminal amino acid of the following peptide - Met-Ile-Val-Glu-Gln-Tyr-Phe-Trp-Asp?
    
11. Which amino acid in the following peptide sequence -- Met-Ile-Leu-Glu-Gly-Gln-Tyr-Phe-Trp-Asp -- is the most flexible in terms of sterically allowed conformations?
    
12. Which amino acid is the least flexible in terms of sterically allowed conformations?
    
13. In the following peptide sequence -- C-D-V-N-Q-P-F-W-E -- the R-group of which amino acid is likely to be the most chemically reactive at physiological pH = 7.0?
    
14. Which standard amino acid is chemically most related to the "mystery molecule" shown here?
    
15. Is the "mystery molecule" shown here a standard amino acid? If you think so, click the button above its correct one-letter designation. If you think NOT, select the button under the label: "This is a non-standard amino acid."
    

EU name: AAHYD0

You can use at a very rough rule-of-thumb that the hydrophilicity of a side
chain is determined by the number of  N and O  atoms in the side chain. The
exception is the N in tryptophan that is almost entirely hydrophobic.
 
Charged N and O atoms are slightly more hydrophilic  than non-charged ones.
And given an equal number of N and O atoms the one with the most carbons or
sulphurs is the more hydrophobic one.
Obviously, this rule-of-thumb is very crude. And further down this page we will
see that it is probably impossible to even make a good rule for hydrophobicity
determination of amino acids. However, as hydrophobicity certainly is the most
important characteristic of the amino acid, everything that helps you work
with in in your head helps you become a better bioinformatician.

Question 44: Use the rule-of-thumb that is boxed above this question (on the website not in this handout...), and look at the sheet with amino acid structure formulas to find 10 pairs of amino acids that look very much like each other, actually they should look so much like each other that they differ by no more than one atom. You can start with Ala-Ser and Thr-Ser, but the rest you have to find yourself. In each case write the more hydrophobic residue of the pair to the left.

Question 45: The scorings matrix used by BLAST reflects how much residues look like each other. If you had to design a scorings-matrix for BLAST, would you give the pairs you listed in the previous question hsigh points or low points?
And where would you put the highest points in your matrix?

Obviously, the rule-of-thumb is just that, a rule-of-thumb. In reality you should determine the pKa of each titratable group, take into account the pH, the salt concentration (and the type(s) of salt involved), the temperature, etcetera. But for now, the rule-of-thumb is good enough.

Question 46: Given the rules of thumbs, and your pairwise sorting, make a list of all amino acids and sort them by hydrophobicity. Start with Trp as the most hydrophobic one at the far left, and end with Arg as the most hydrophilic one at the far right.
Discuss the answer with an assistant before continuing with the subsequent questions.

Question 47: Look up the pKa values of the titratable groups ( http://swift.cmbi.umcn.nl/teach/aainfo/index.shtml; section 3.3).
Why are the pKa values given for only 7 amino acids?
What are the pKa values of the two positive residues? And of the two negative values? And what is the pH in the cytosol of a human cell? And why are these questions combined in one question; in other words what is the deeper meaning here?
The pKa of Cys is ~9.3. Does that make Cys a basic residue?
Now go back to the hydrophobicity scales and think about what the assistants told you about the position of Lys. Does that story now make sense?

EU name: AAHYD

Hydrophobicity scales

You are not the only ones who have made a hydrophobicity scale. Others have done that many years ago already. And many scales were made with very sophisticated techniques.

Look up the hydrophobicity scales ( http://swift.cmbi.umcn.nl/teach/aainfo/hydro.shtml) and first find out what this is all about; what is in those scales; how were they determined; etc.

Question 48: Look at the scales mentioned above (on the website, not in the handout) and answer:
Which is the most hydrophobic residue?
What is wrong with the previous question?
What is the difference between scales 1&4 versus 2&3? In other words, which experiments were done to arrive at the values in these scales?
Explain the different position for cysteine in 1&4 versus 2&3.
Explain the position of proline in the scales. Is it located where you expected it?
Explain the positions of arginine en lysine in the different scales.
Can you think of a possible reason that arginine and lysine are hardly ever found inside a protein, while glutamic acid and aspartic acid can occasionally be found there?

Accessibility

There is, of course, a strong relation between hydrophobicity and the preference of amino acids for the surface or the inside of the protein. But this is a preference, and not a law. Hydrophobic residues can be found at the surface and in the core of the protein, whereas hydrophilic ones tend to be found much more often at the surface than inside the protein.

You often see in standard text books that hydrophobic residues sit in the core,
and hydrophilic ones at the surface of proteins. In the next step of this
course you can see for yourself that that is not true.

Look up the page about solvent accessible surfaces ( http://swift.cmbi.umcn.nl/teach/aainfo/access.shtml) Note: One water molecule has a diameter of about 2.8 Ångström  and it can touch a surface of about 10 Ångström²  of the surface of a protein. A SEA of 30 Ångström²  means that about three water molecules can touch this amino acid (on average in the dataset used by the bioinformaticians who produced this table).

Question 49: Look up the above scales (on the website, not in the handout) and answer the (simple) questions:
Which amino acids will be more accessible to solvent (water), those at the exterior (outside) or those in the interior (inside) of the protein?
Describe in your own words the meaning of the numbers in the table.
(Just a small side-step: How do you think these numbers are being calculated? In other words, what does the computer algorithm look like)?
Which amino acids are on average most accessible?
Which amino acid is on average least accessible?
What is so dangerous about questions like the previous two?
Try to explain why methionine is relatively often found at the outside of the protein.
Proline has an unexpected SEA distribution. Can you explain why?

When thinking about residue hydrophobicity and the surface location of proteins there is a nice, for many people surprising, realisation.

Question 50: If you observed a polar residue in the core of a protein, and you mutate it into a hydrophobic residue, the protein often gets more stable. But when you observe a hydrophobic residue at the surface and mutate it into a polar one, you normally don't see any effect on the stability.
Think about the entropy of water. Realize that stability is measured as the temperature at which the protein folding process (folded -- unfolded is just in equilibrium.
And now the question: Why is this?

Amino acid structure

Question 51: Draw the general structure of one amino acid (use R to indicate the side chain). Indicate the N-terminal side and the C-terminal side of the amino acid. Also put the names of the backbone atoms in the drawing (N, Cα, C, O).
Draw a dipeptide Val-Lys, (that is two amino acids joined by a peptide bond)..
Draw the dipeptide Lys-Val.
Are those dipeptides identical?

Hydrogen bonds

Question 52: Which atoms in the protein backbone can form hydrogen bonds?.
Which backbone atom donates the hydrogen atom (hydrogen bond donor)?.
Which backbone atom accepts the hydrogen atom (hydrogen bond acceptor)?.
And, actually, now that we are talking about it: What is a hydrogen bond?.

Alignments

Question 53: The main reason for our study of the amino acids is understanding them well enough to interpret (and if needed correct) alignments. So, lets take a look at some alignments. It should be possible to answer these questions using no more than a bit of brainpower (to think about the relative importance of residues) and the information provided in the amino acid information pages. For each pair of alignments listed below, determine which is the best one (left or right) and explain why.

1
LLLWASLLL  LLLWASLLL
LLLLWSLLL  LLLWSLLLL

2
LLLCANLLL  LLLCANLLL
LLLLCNLLL  LLLCNLLLL

3
LLLRAGLLL  LLLRAGLLL
LLLLRGLLL  LLLRGLLLL

4
LLLHANLLL  LLLHANLLL
LLLLHNLLL  LLLHNLLLL

5
LLLPAKLLL  LLLPAKLLL
LLLLPKLLL  LLLPKLLLL

And now, as the grande-finali, the (in)famous amino acid test

Question 54: For each amino acid, list its 1-letter code, its 3-letter code, its full name, whether it is hydrophobic/hydrophilic/inbetween, its size (small/medium/large), its charge at pH 6 a 7, its secondary structure, and anything special you know about this amino acid.
Feel free to ask the assistants for an empty amino acid sheet to make this exercise administratively easier. (There is an extra copy for you to print under the heading 'test exams').

Question 55: Go through the amino acid seminar and figure out what are special characteristics of the amino acids. Try to summarise these characteristics in 1 word per characteristic otherwise it won't fit in the small box.