Shellular biology – exploring the biochemistry and physiology of a protein nanocompartment
- Author(s): Cassidy-Amstutz, Caleb
- Advisor(s): Savage, David F
- et al.
Bacteria and Archaea use proteinaceous compartments for a variety of different reasons such as creating unique chemical environments or isolating pathways with toxic intermediates. Recently, a new class of protein compartment was discovered and named for its founding member, encapsulin. Encapsulins are structural similar to bacterial microcompartments but are smaller; hence, encapsulins are interchangeably called nanocompartments. Encapsulins assemble into 24-32 nm icosahedral shells and usually package a single type of cargo protein such as a ferritin-like protein or a peroxidase. Cargo proteins are directed to the interior of encapsulins through an interaction between the encapsulin lumen and the C-terminus of the cargo protein. With the recent discovery of encapsulins, there are many fundamental questions to answer in regards to both the biochemistry and physiology of nanocompartments. In this work, I began by heterologously expressing and then purifying the T. maritima encapsulin. I was able to show the subunits readily and robustly assemble into nanocompartments and these nanocompartments are resistant to both thermal and chemical insults. Through fusion of the C-terminus from a cargo protein to GFP, I was able to load encapsulins with GFP both in vitro and in vivo. However, in vitro loading was only accomplished by disassembling and reassembling encapsulin in the presence of targeting, suggesting that in vivo encapsulins are only competent to load cargo during assembly. While purifying T. maritima encapsulin, I discovered the shell is a flavoprotein that co-purifies with riboflavin and flavin mononucleotide. Cryo-electron microscopy identified the flavin binding site as being adjacent to a surface tryptophan and mutation of the tryptophan to alanine substantially reduced the amount of flavin that co-purified with encapsulins. Flavins are common cellular electron transporters and the presence of a flavin tightly bound to an encapsulin shell suggests it may be related to the functioning of the native cargo, a ferritin-like protein that oxidizes ferrous iron and forms an iron oxide mineral. I am currently investigating whether the bound flavins act as electron donors or acceptors for the reduction or oxidation of iron. This work was one of the first biophysical characterizations of an encapsulin and the first suggestion that nanocompartments may not be passive barriers separating the encapsulin interior from the cytosol. Instead, encapsulins may play a direct and active role in the function of encapsulated cargo.