Kenneth J. Woycechowsky

Directed Evolution of a Protein Container

An engineered protein container protects its bacterial host by efficient and selective encapsulation of a toxic protease. Confinement of enzymes in protein nanocompartments represents a potentially powerful strategy for controlling catalytic activity in cells. By using a simple electrostatically based tagging system for protein encapsulation, we successfully sequestered HIV protease, a toxic enzyme when produced cytoplasmically, within an engineered lumazine synthase capsid. The growth advantage resulting from protecting the Escherichia coli host from the protease enabled directed evolution of improved capsids. After four rounds of mutagenesis and selection, we obtained a variant with a 5- to 10-fold higher loading capacity than the starting capsid, which permitted efficient growth even at high intracellular concentrations of HIV protease. The superior properties of the evolved capsid can be ascribed to multiple mutations that increase the net negative charge on its luminal surface and thereby enhance engineered Coulombic interactions between host and guest. Such structures could be used for diverse biotechnological applications in living cells.

Native Disulfide Bond Formation in Proteins

Native disulfide bond formation is critical for the proper folding of many proteins. Recent studies using newly identified protein oxidants, folding catalysts, and mutant cells provide insight into the mechanism of oxidative protein folding in vivo. This insight promises new strategies for more efficient protein production.

Catalysis of Oxidative Protein Folding by Small-Molecule Diselenides†

The production of recombinant, disulfide-containing proteins often requires oxidative folding in vitro. Here, we show that diselenides, such as selenoglutathione, catalyze oxidative protein folding by O 2. Substantially lower concentrations of a redox buffer composed of selenoglutathione and the thiol form of glutathione can consequently be used to achieve the same rate and yield of folding as a standard glutathione redox buffer. Further, the low p K a of selenols extends the pH range for folding by selenoglutathione to acidic conditions, where glutathione is inactive. Harnessing the catalytic power of diselenides may thus pave the way for more efficient oxidative protein folding.

Diselenides as universal oxidative folding catalysts of diverse proteins

Small-molecule diselenides show considerable potential as catalysts of oxidative protein folding. To explore their scope, diselenide-containing redox buffers were used to promote the folding of proteins that varied in properties such as size, overall tertiary structure, number of disulfide bonds, pI value, and difficulty of in vitro folding. Diselenides are able to catalyze the oxidative folding of all proteins tested, providing significant increases in both rate and yield relative to analogous disulfides. Compared to the disulfide-linked dimer of glutathione (the most commonly used oxidant for in vitro protein folding), selenoglutathione provided markedly improved efficiencies in the folding of biotechnologically important proteins such as hirudin, lysozyme, human epidermal growth factor and interferon α-2a. Selenoglutathione also enhances the renaturation of more challenging targets such as bovine serum albumin, whose native state contains 17 disulfide bonds, and the Fab fragment of an antibody. In the latter case, micromolar amounts of selenoglutathione are able to match the modest yield provided by a previously optimized redox buffer, which contains millimolar levels of glutathione. Taken together, the folding reactions of these diverse proteins exemplify the advantages and limitations of diselenide catalysts.

Reagentless oxidative folding of disulfide-rich peptides catalyzed by an intramolecular diselenide.

In cysteine-rich peptides, diselenides can be used as a proxy for disulfide bridges, since the energetic preference for diselenide bonding over mixed selenium-sulfur bonds simplifies folding. Herein we report that an intramolecular diselenide bond efficiently catalyzes the oxidative folding of selenopeptide analogs of conotoxins, and serves as a reagentless method to substantially accelerate formation of various native disulfide bridging patterns.

Relative Tolerance of an Enzymatic Molten Globule and Its Thermostable Counterpart to Point Mutation

Enzyme structures reflect the complex interplay between the free energy of unfolding (DeltaG) and catalytic efficiency. Consequently, the effects of point mutations on structure, stability, and function are difficult to predict. It has been proposed that the mutational robustness of homologous enzymes correlates with a higher initial DeltaG. To examine this issue, we compared the tolerance of a natural thermostable chorismate mutase and an engineered molten globular variant to targeted mutation. These mutases possess similar sequence, structure, and catalytic efficiency but dramatically different DeltaG values. We find that analogous point mutations can have widely divergent effects on catalytic activity in these scaffolds. In a set of five rationally designed single-amino acid changes, the thermostable scaffold suffers activity losses ranging from 50-fold smaller, for an aspartate-to-glycine substitution at the active site, to 2-fold greater, for a phenylalanine-to-tryptophan substitution in the hydrophobic core, versus that of the molten globular scaffold. However, biophysical characterization indicates that the variations in catalytic efficiency are not caused by losses of either secondary structural integrity or thermodynamic stability. Rather, the activity differences between variant pairs are very much context-dependent and likely stem from subtle changes in the fine structure of the active site. Thus, in many cases, it may be more productive to focus on changes in local conformation than on global stability when attempting to understand and predict how enzymes respond to point mutations.

Catalysis of Protein Folding by an Immobilized Small-Molecule Dithiol

The isomerization of non‐native disulfide bonds often limits the rate of protein folding. Small‐molecule dithiols can catalyze this process. Here, a symmetric trithiol, tris(2‐mercaptoacetamidoethyl)amine, is designed on the basis of criteria known to be important for efficient catalysis of oxidative protein folding. The trithiol is synthesized and attached to two distinct solid supports via one of its three sulfhydryl groups. The resulting immobilized dithiol has an apparent disulfide E°′ = –208 mV, which is close to that of protein disulfide isomerase (E°′ = –180 mV). Incubation of the dithiol immobilized on a TentaGel resin with a protein containing non‐native disulfide bonds produced only a 2‐fold increase in native protein. This dithiol appeared to be inaccessible to protein. In contrast, incubation of the dithiol immobilized on styrene‐glycidyl methacrylate microspheres with the non‐native protein produced a 17‐fold increase in native protein. This increase was 1.5‐fold greater than that of a monothiol immobilized on the microspheres. Thus, the choice of both the solid support and thiol can affect catalysis of protein folding. The use of dithiol‐decorated microspheres is an effective new strategy for preparative protein folding in vitro.

Small-molecule diselenides catalyze oxidative protein folding in vivo.

Prokaryotic cells normally rely on periplasmic oxidoreductases to promote oxidative protein folding. Here we show that simple diselenides can also facilitate the conversion of dithiols to disulfides in vivo, functionally replacing one such oxidoreductase, DsbA, in the oxidative folding of diverse proteins. Structurally analogous disulfides provide no detectable effect when used at concentrations that gave optimal activity with diselenides, and even at 100- to 1000-fold higher levels they show only partial activity. The low concentrations of diselenides needed to fully negate typical DsbA knockout phenotypes suggest catalysis in vivo, a property that sets these additives apart from other small molecules used in chemical biology. Supplementing growth media with cell-permeable organocatalysts provides a potentially general and operationally simple means of fine-tuning the cellular redox environment.

In Vivo 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 Aquifex aeolicus (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 in vivo. 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.

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 (LSs) 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 LSs 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.