Chemists are fascinated by the cell’s ability to compartmentalize, and they have been trying to mimic it for years. Separate things like the nucleus, mitochondria, and other compartments keep otherwise incompatible biochemical processes functioning cohesively in one cell. One of the simplest compartments is a protein shell, like the capsid of a virus. Protein shells are usually highly organized structures with defined geometric shapes and sizes. Scientists have altered natural protein shells for a variety of purposes, from crystallizing inorganic nanoparticles to the delivery of drugs. In a new paper, they've engineered a capsule that can neutralize a toxic protein.
Bigna Wörsdörfer, Kenneth Woycechowsky, and Donald Hilvert used directed evolution, where they specifically select for certain traits in a protein, to engineer a container that is able to control the catalytic activity of an enzyme called a protease from the HIV genome. Their capsule was made from a protein called lumazine synthase, found in the bacteria Aquifex aeolicus (AaLS). AaLS is an enzyme that is involved in the biosynthesis of riboflavin (vitamin B2); it normally forms icosahedral-shaped capsids made from either 60 or 180 identical subunits.
Scientists can modify AaLS capsids to grab guest molecules based on simple electrostatic interactions, the attraction between positive and negative charges. They do this by changing four amino acids in each subunit of the capsid—they switched out four positively charged arginines for the negatively charged glutamates. This adds a total of 240 or 720 negative charges, depending on if a capsid has 60 or 180 subunits. The authors called the negatively charged capsids AaLS-neg.
AaLS-neg prefers to grab positively charged proteins, meaning that researchers can target a protein for encapsulation by adding a tag of 10 positively charged arginines at the end of a target protein. The deca-arginine tag adds a lot of positive charge, which is attracted to AaLS-neg.
For this work, the authors placed deca-arginine tags on HIV protease, a protein cutting enzyme that is essential for the HIV life-cycle. Normally, HIV protease is toxic to E. coli, presumably because it cuts essential proteins. If HIV protease and AaLS-neg are coproduced in E. coli, AaLS-neg should encapsulate HIV protease and the cells should be saved from the toxic effects.
The authors induced the growth of HIV protease along with AaLS-neg and found that AaLS-neg does rescue cell growth, but not at high concentrations of protease. Nevertheless, coproduction of AaLS-neg and HIV protease in E. coli cells produces a selectable trait, namely survival. The researchers put random mutations in the gene that encodes for AaLS-neg, then selected for surviving cells in the presence of ever-higher concentrations of protease.
After four rounds of selection, the researchers produced AaLS-13, a variant of the original AaLS-neg that has over a 5-fold greater loading capacity for HIV protease. Cells with AaLS-13 can survive in high concentrations of the toxic enzyme. AaLS-13 has mutations that give it even more negative charge, making it better at grabbing its positively charged cargo.
However, the encapsulation only occurs during the assembly of the AaLS-13 capsid. When the authors mixed already-assembled AaLS-13 with HIV protease, the two did not form a complex.
The researchers propose that their system, comprised of a protein capsid and a matching arginine tagged target enzyme, is a viable method of regulating enzyme activity within a cell. Their design is simple and easily applicable to other proteins.
But the system has selectivity issues, as the authors noted that AaLS-13 will encapsulate other positively charged proteins when there isn't HIV protease around. In the long term, it might be better to start with a smaller charge difference and then do more rounds of selection, which might allow the capsule to evolve specificity for some specific features of its target protein.
Listing image by NASA