PLN Guide

PLN stands for Protein Line Notation, the native format used by the Proteax software to define peptide and protein structures. It is a line entry format that covers plain linear and cyclic sequences, multiple chains, and bridged and crosslinked residues and terminals.

In its simplest form a PLN entry is a string of uppercase single-letter residue codes with standard H- and -OH terminals added at the ends. An example of this would be the first chain of human insulin as shown below.

Displaying this graphically as sequence and structure:

To put multiple chains into a PLN entry, the chains should be separated by periods. So to add the second chain of human insulin, add a period and then the second chain.

We now have the full sequence of human insulin. The sequence graphics uses alternating green and blue background colors to tell chains apart.

This is not quite the real structure of human insulin. We need to add disulfide bridges.

A disulfide bridge is the simplest crosslink feature in PLN. It creates a disulfide bond linking two reactive sulfur atoms. As seleniumcysteine is a commonly occurring residue PLN accepts that disulfide bridges form between both sulfur and selenium atoms.

A disulfide bridge is defined in PLN by adding a unique number in parentheses after the residue or terminal that the disulfide bond should form to. Proteax will automatically find the reactive sulfur or selenium in the residue or terminal structure and create the disulfide bond.

Human insulin has three disulfide bridges and here we number them 1 - 3. The numbers are used to pair up the residues/terminals that should be linked with disulfide bonds.

We now have an accurate structural definition of human insulin.

The numbers used do not carry any meaning but are solely used to uniquely link pairs of residues or terminals with disulfide bonds. We could use any pairs of numbers, so the following PLN will express the exact same sequence and structure as above.

A lactam cycle links a reactive nitrogen with an acid group, displacing a water molecule as shown below.

Lactam cycles are specified in PLN much like disulfide bridges. In the parentheses, add the keyword "lactam" or "cyclo" in front of the unique bridge number. An example is shown below.

The graphics of the structure has been switched to condensed mode so only structurally modified or crosslinked residues are shown as full structure. Standard residues are shown as "bubbles".

A terminal may also be the endpoint of a lactam cycle. Standard N-terminals provide a reactive amino group while C-terminals provide an acid group. The following two peptide examples demonstrate this. Note how the "(cyclo)" keyword replaces the "H-" or "-OH" standard terminal when the terminal is part of a bridge.

Note in the last example that when you cyclize a chain to be fully cyclic you do not need to add a numerical identifier after the "cyclo" keyword. You are still allowed to do it, but it is not necessary.

Finally, an example of linking two chains from a residue to a terminal, yielding a branched peptide.

Another reactive crosslink formation is thioether bridges. This is performed via the "thio" keyword and builds an S-C link between a reactive sulfur site and a C-OH group (displacing the -OH).

A simple example with a single thioether bridge.

Thioether bridges can be used in PLN all the same places as lactam bridges, provided that the linked residues or terminals have reactive sites that match the thioether chemistry.

When a peptide is post-translationally or chemically modified, the standard one-letter residue codes and terminals do not faithfully represent the actual structure. In those cases, PLN annotations let you replace the standard residue codes and terminals with custom structures.

Custom structures that are used often will be stored in the Proteax modification database. Your modification database may however not have the structures used in the examples below, and that will make the PLN unreadable at your end. All PLN examples can therefore be found in self-contained format using inline modifications at the end of this document. This will let you copy/paste the PLN directly without having to register any additional custom structures in your modification database.

Bridges can be formed to modified residues and terminals, just like standard residues and terminals. Place the bridge parentheses after the modification name, like you would place it after a residue code or in place of a standard terminal.

In this section an "S-term" N-terminal structure containing a sulfur is used.

The sulfur of the terminal structure can be linked to a cysteine in the sequence through a normal disulfide bridge as in the example below.

Some complex structures cannot be expressed using ordinary sidechain modifications and standard reactive bridges. You may have sidechains with ambiguous reactive sites, multiple residues that are linked (three or more), or simply linkages that happen via other reactions than the three standard bridges that Proteax supports.

In that case you will want to use generic crosslinks.

Residue extensions let you attach terminal structures to residue sidechains. This way you can combine existing structures without having to register new residue structures.

The general format of a residue extension is

where

An example is biotinylation of lysine. This can be achieved by extending a standard lysine structure with an N-terminal biotin. These two structures are shown below.

A sequence that generates a lysine extended with a biotinylation can then be:

The optional terminal transforms can be used to flip the terminal structure’s stereochemistry (the "d" transform) or change the terminal structure’s layout (the remaining options). An example is flipping the horizontal layout of the biotin with the "flipHorz" transform.

We can also flip the terminal vertically and rotate it 15 degrees.

To rotate clockwise, use negative numbers e.g. "rotate-15".

Finally, an example of biotinylation of a modified lysine, N6-methyllysine.

If you copy the below PLN examples from a PDF file you are unfortunately likely to see errors when pasting the text into the PLN editor. Some PDF readers convert regular spaces into special non-breaking-space characters and others remove hyphens from the end of a line. If you experience these issues, you can copy from the HTML version instead (use the second link to go straight to this section):

http://biochemfusion.com/doc/PLN_Guide/PLN_Guide.html
http://biochemfusion.com/doc/PLN_Guide/PLN_Guide.html#_pln_for_copying

[Gla] usage.

[Gla] usage with amidated C-terminal.

[Gla] D-forms.

N-terminal biotinylation.

N-terminal with sulfur.

Crosslinking in a stapled peptide.

N-terminally-linked crosslink.

Crosslink with a core.

Crosslink with a core - endpoint (a) stereo-inverted.

Residue extension - Lys with N-terminal biotin.

Residue extension - N6-methyllysine with N-terminal biotin.