Conversion of a Dodecahedral Protein Capsid into Pentamers via Minimal Point Mutations

Abstract

Protein self-assembly relies upon the formation of stabilizing noncovalent interactions across subunit interfaces. Identifying the determinants of self-assembly is crucial for understanding structure-function relationships in symmetric protein complexes and for engineering responsive nanoscale architectures for applications in medicine and biotechnology. Lumazine synthases (LS's) comprise a protein family that forms diverse quaternary structures, including pentamers and 60-subunit dodecahedral capsids. To improve our understanding of the basis for this difference in assembly, we attempted to convert the capsid-forming LS from Aquifex aeolicus (AaLS) into pentamers through a small number of rationally designed amino acid substitutions. Our mutations targeted side chains at ionic (R40), hydrogen bonding (H41), and hydrophobic (L121 and I125) interaction sites along the interfaces between pentamers. We found that substitutions at two or three of these positions could reliably generate pentameric variants of AaLS. Biophysical characterization indicates that this quaternary structure change is not accompanied by substantial changes in secondary or tertiary structure. Interestingly, previous homology-based studies of the assembly determinants in LS's had identified only one of these four positions. The ability to control assembly state in protein capsids such as AaLS could aid efforts in the development of new systems for drug delivery, biocatalysis, or materials synthesis.

Diverse Bacterial Microcompartment Organelles

“…Pathway encapsulation would not only increase flux through pathways with poor kinetic properties but also allow control of molecules that enter or exit, sequestration of intermediates, and optimization of the local chemical microenvironment. Several possible compartment platforms have been suggested for both in vivo and in vitro applications, including viral capsids, lumazine synthase capsids, polymeric cages, liposomes, and bacterial MCPs (184)(185)(186)(187). Bacterial MCPs seem particularly well suited for the development of intracellular bioreactors, since evolution has designed MCPs to isolate biochemical reactions with the purpose of regulating enzyme activity, enhancing pathway flux, and protecting cells from toxic intermediates.…”

Reversible loading of thiol-modified curcumin in an engineered protein capsid

“…In the rst round, the naturally occurring cysteine at position 37 was changed to alanine. For this mutagenesis reaction, the plasmid pMG-AaLS-noHis 27 was used as the template and the primers GT_C37AF and GT_C37AR (5 0 -GTGGAGGGTGCAATTGATGCTATAGTCCG-TCATGGCGGC-3 0 and 5 0 -GCCGCCATGACGGACTATAGCATCAA-TTGCACCCTCCAC-3 0 , respectively, mutations indicated in bold) were used to introduce the C37A mutation, yielding plasmid pMG-AaLS-C37A. In a second round of site-directed mutagenesis, glutamate at position 122 was replaced by cysteine.…”

“…The resulting plasmid aer both rounds of mutagenesis, pMG-AaLS-IC, encodes the double variant C37A/E122C-AaLS (AaLS-IC). For both mutations, the mutagenesis PCR was carried out similarly to a previously reported procedure 27 and involved 18 cycles of denaturation (95 C for 30 s), annealing (58 C for 30 s), and primer extension (72 C for 6 min). The coding portions of the resulting plasmids (pMG-AaLS-C37A and pMG-AaLS-IC) were conrmed by DNA sequencing (University of Utah DNA sequencing core facility).…”

“…AaLS-IC capsids were produced in Escherichia coli BL21 (DE3) cells from plasmid pMG-AaLS-IC and puried using a combination of ammonium sulfate treatment, anion exchange chromatography, and size-exclusion chromatography, according to a previously published procedure. 27 The purity of each protein was conrmed by SDS-PAGE. The yield was determined by Bradford assay.…”

“…1A) is built from 60 identical subunits, 26 which assemble as a dodecamer-ofpentamers. 27 A central tunnel, which is $0.9 nm wide at its narrowest point, runs through each of the pentameric building blocks, and potentially provides a means for small molecules to diffuse across the capsid shell. 28 Compared with viral nanoparticles, the AaLS capsid is relatively small, with an outer diameter of 16 nm and an inner diameter of 9 nm.…”