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Lysine is the only canonical amino acid, which bears an amino group. This is a must-have moiety when designing proteins, for example, as a site for ligations. Strong covalent ligations can be made via an amide bond with a carboxyl-group reagent, likewise in biotin attachment forming biocitin. More loose attachment can be made via a Schiff-base with an aldehyde or ketone counterpart, for instance, when pyridoxal-5-phosphate is harbored by an enzyme the aldehyde group is attached to a lysine amino-group. Attachment of a carbon dioxide molecule creates carboxy-lysine, an important general base in enzymatic transformations.

No wonder that lysine (along with cysteine) is the most common target for man-made so-called ‘bioorthogonal’ attachment of various molecules. Cross-linking mass spectrometry, a method to track interacting protein-protein interactions, relies primarily on covalent cross-linking of lysine residues at interacting interfaces.

There is an ample amount of other lysine-specific chemical aspects. For example, lysine is involved in numerous catalytic triads promoting protonation-deprotonation, nucleophilic mechanisms and others. There is a rich post-translational modification chemistry done with this side chain, for instance, in lysine-rich DNA-packing histone proteins.

But let’s discuss one interesting chemical aspect of the lysine, its length. Why is it so important that lysine has four methylene units between the amino-group and the backbone?

Let’s see whether the linker can be shorter. First, we can exclude diamino-butyric acid (Dab) from occurrence in translation process due to important chemical reasons. This amino acid, when attached onto a tRNA in a form of an amino acyl-tRNA ester would most likely form intramolecular cyclization product, thus, it would cleave itself and then leave in the form of an amino-pyrrolidone. The anomaly of 5-membered rings is very familiar in organic chemistry, and basically, whenever a nucleophilic reaction leads to a 5-membered ring, the likelihood of its occurrence becomes enormous. The same reason explains why homoserine translation would also be impossible.

What if linker is just one carbon, and the amino acid is diaminopropionic acid (Dap). Here a positively-charged ammonium group would be placed very close to the backbone. However, the problem is that this group would require an extensive solvation by water. Thus, solvation shell would most likely interfere with the neighboring residues in a context of a compact helical structure, such as α-helix. Another outcome, is that surrounded by other side chains, the ammonium group would hardly be accessible for chemical reactions, such as Schiff-base formation and others. To make it more accessible, yet well solvated, one should elongate the linker to allow maximal emergence of the terminal nitrogen atom into the solvent. Like a toy balloon or a kite. The higher it rises, the more solvated and accessible it becomes.

How high can it go? Remember, that the linker in an olygomethylene (CH2)n, and this is a hydrophobic chain. We cannot increase its length forever, because at some point it would start to collapse with the neighboring hydrophobic side chains and with itself. Therefore, four methylene units is probably the optimal distance at which these different considerations find a compromise. Actually, people often forget that lysine side-chain is quite hydrophobic due to its length, and this is the ammonium charge, which makes it hydrophilic eventually. However, when the charge is eliminated by N-acetylation or in N-Boc lysine, the amino acid can become fairly hydrophobic.

Interesting readings:

- Padmanabhan, S. et al. Helix propensities of basic amino acids increase with the length of the side-chain. J. Mol. Biol., 257, 1996, 726-734, doi: 10.1006/jmbi.1996.0197

The paper addressed the helix propensity of lysine homologs in a context of Lys(Ala)4-based repetitive peptide structures.

- Rappsilber, J. The beginning of a beautiful friendship: Cross-linking/mass spectrometry and modelling of proteins and multi-protein complexes. J. Struct. Biol., 173, 2011, 530-540, doi: 10.1016/j.jsb.2010.10.014

The perspective explains how tethering interacting surfaces together can help to identify new protein-protein interaction networks. Lysine is the key amino acid, since it’s abundant on the surface, and can be easily ligated and cross-linked.

- Koniev, O. and Wagner, A. Developments and recent advancements in the field of endogenous amino acid selective bond forming reactions for bioconjugation. Chem. Soc. Rev., 44, 2015, 5495-5551, doi: 10.1039/C5CS00048C

A recent review on biorthogonal chemistry on proteins. Lysine ligations are regarded as “the oldest and most versatile techniques for protein conjugation”.

- Maier, G. P. et al. BIOLOGICAL ADHESIVES. Adaptive synergy between catechol and lysine promotes wet adhesion by surface salt displacement. Science, 349, 2015, 628-632, doi: 10.1126/science.aab0556

How to make a good adhesive? The paper demonstrates that combination of a lysine positive charge and a catechol leads to a dramatic synergetic enhancement of adhesive properties.