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Solving The Pathway of LADDERane biosynthesis

Periodic Reporting for period 4 - STePLADDER (Solving The Pathway of LADDERane biosynthesis)

Reporting period: 2021-12-01 to 2023-05-31

Lipids are the main molecules that make up cell membranes, i.e. they form the enclosure of a cell. Typically, they contain long linear chains of carbon atoms, which are relatively easy for the cell to produce, but many organisms make interesting modifications to this basic design to suit their purposes. Arguably, the most interesting and surprising among these modifications is the incorporation of so-called “ladderanes”, which is seen in the lipids of several bacteria. Ladderanes are groups of carbon atoms arranged as a series of interconnected squares, reminiscent of a ladder (see figure 1). This is an extremely unusual arrangement for carbon atoms and is very difficult to make. The STePLADDER project investigates the mechanisms used by cells to make such molecules, with a view to expanding our own abilities to synthesize complicated molecules in the lab This is important for medicine, nanotechnology and many other fields.
To find out how ladderanes are made, we are combining methods from various fields of science: molecular biology to obtain the proteins responsible for producing ladderanes, biophysics to study their structure, biochemistry to study them inside the cell, and organic chemistry to produce and investigate various lipid molecules.
We began by establishing the various methods needed for the project. After production of several interesting lipid-synthesizing proteins from ladderane-producing bacteria, we set up methods to bind various lipids to them, and for their analysis. This gave us insight in several of the proteins' structures, and into how they work. Moreover, we have been studying the production of ladderanes inside living cells. All of these results will be combined to yield insight into how ladderanes are produced.
Importantly, we developed new methods to obtain proteins directly from ladderane-producing organisms, which are notoriously difficult to work with in the lab. In addition, we adapted various biochemical methods for use with these organisms and their proteins.
Using biophysical methods, we have determined the structures of several of these proteins to a level of detail where we can see how their individual atoms are arranged. This allows us to precisely determine how lipid molecules are bound and modified by these proteins. Among these is the structure of a “complex” of two proteins and a lipid, which shows how the two proteins work together to modify the lipid. Moreover, unexpectedly we found that two other proteins, which were thought to work separately, form a tightly intertwined "complex" with one another, where one protein probably regulates the function of the other. Moreover, of yet another protein we now know the function.
Figure 1. A ladderane molecule. Carbon atoms are black, hydrogen atoms white.