A small-molecule catalyst of protein folding in vitro and in vivo

AbstractBackground: The formation of native disulfide bonds between cysteine residues often limits the rate and yield of protein folding. The enzyme protein disulfide isomerase (PDI) catalyzes the interchange of disulfide bonds in substrate proteins. The two -Cys-Gly-His-Cys- active sites of PDI provide a thiol that has a low pKa value and a disulfide bond of high reduction potential (E°').Results: A synthetic small-molecule dithiol, (±)-trans-1,2-bis(2-mercaptoacetamido)cyclohexane (BMC), has a pKa value of 8.3 and an E°' value of −0.24 V. These values are similar to those of the PDI active sites. BMC catalyzes the activation of scrambled ribonuclease A, an inactive enzyme with non-native disulfide bonds, and doubles the yield of active enzyme. A monothiol analog of BMC, N-methylmercaptoacetamide, is a less efficient catalyst than BMC. BMC in the growth medium of Saccharomyces cerevisiae cells increases by > threefold the heterologous secretion of Schizosaccharomyces pombe acid phosphatase, which has eight disulfide bonds. This effect is similar to that from the overproduction of PDI in the S. cerevisiae cells, indicating that BMC, like PDI, can catalyze protein folding in vivo.Conclusions: A small-molecule dithiol with a low thiol pKa value and high disulfide E°' value can mimic PDI by catalyzing the formation of native disulfide bonds in proteins, both in vitro and in vivo

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

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 <i>Aquifex aeolicus</i> (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

Encapsulation and Controlled Release of Protein Guests by the <i>Bacillus subtilis</i> Lumazine Synthase Capsid

In <i>Bacillus subtilis</i>, the 60-subunit dodecahedral capsid formed by lumazine synthase (BsLS) acts as a container for trimeric riboflavin synthase (BsRS). To test whether the C-terminal sequence of BsRS is responsible for its encapsulation by BsLS, the green fluorescent protein (GFP) was fused to either the last 11 or the last 32 amino acids of BsRS, yielding variant GFP11 or GFP32, respectively. After purification, BsLS capsids that had been co-produced in bacteria with GFP11 and GFP32 are 15- and 6-fold more fluorescent, respectively, than BsLS co-produced with GFP lacking any BsRS fragment, indicating complex formation. Enzyme-linked immunosorbent assay experiments confirm that GFP11 is localized within the BsLS capsid. In addition, fusing the last 11 amino acids of BsRS to the C-terminus of the Abrin A chain also led to its encapsulation by BsLS at a level similar to that of GFP11. Together, these results demonstrate that the C-terminal tail of BsRS can act as an encapsulation tag capable of targeting other proteins to the BsLS capsid interior. As with the natural BsLS–BsRS complex, mild changes in pH and buffer identity trigger dissociation of the GFP11 guest, accompanied by a substantial expansion of the BsLS capsid. This system for protein encapsulation and release provides a novel tool for bionanotechnology

<i>In Vivo</i> Encapsulation of Nucleic Acids Using an Engineered Nonviral Protein Capsid

In Nature, protein capsids function as molecular containers for a wide variety of molecular cargoes. Such containers have great potential for applications in nanotechnology, which often require encapsulation of non-native guest molecules. Charge complementarity represents a potentially powerful strategy for engineering novel encapsulation systems. In an effort to explore the generality of this approach, we engineered a nonviral, 60-subunit capsid, lumazine synthase from <i>Aquifex aeolicus</i> (AaLS), to act as a container for nucleic acid. Four mutations were introduced per subunit to increase the positive charge at the inner surface of the capsid. Characterization of the mutant (AaLS-pos) revealed that the positive charges lead to the uptake of cellular RNA during production and assembly of the capsid <i>in vivo</i>. Surprisingly, AaLS-pos capsids were found to be enriched with RNA molecules approximately 200–350 bases in length, suggesting that this simple charge complementarity approach to RNA encapsulation leads to both high affinity and a degree of selectivity. The ability to control loading of RNA by tuning the charge at the inner surface of a protein capsid could illuminate aspects of genome recognition by viruses and pave the way for the development of improved RNA delivery systems

A Protein-Capsid-Based System for Cell Delivery of Selenocysteine

Selenocysteine (Sec) has received a lot of attention as a potential anticancer drug. However, its broad cytotoxicity limits its therapeutic usefulness. Thus, Sec is an attractive candidate for targeted drug delivery. Here, we demonstrate for the first time that an engineered version of the capsid formed by Aquifex aeolicus lumazine synthase (AaLS) can act as a nanocarrier for delivery of Sec to cells. Specifically, a previously reported variant of AaLS (AaLS-IC), which contains a single cysteine per subunit that projects into the capsid interior, was modified by reaction with the diselenide dimer of Sec (Sec₂) to generate a selenenylsulfide conjugate between the capsid and Sec (AaLS-IC-Sec). Importantly, it was determined that the structural context of the reactive cysteine was important for efficient capsid loading. Further, the encapsulated Sec could be quantitatively released from AaLS-IC-Sec by reducing agents such as glutathione or dithiothreitol. To assess cellular penetrance capabilities of AaLS-IC-Sec and subsequent cytotoxic response, six different cells line models were examined. Across the cell lines analyzed, cytotoxic sensitivity correlated with cellular uptake and intracellular trafficking patterns. Together these findings suggest that the engineered AaLS-IC capsid is a promising vehicle for targeted cell delivery of Sec