In Vitro and Cellular Self-Assembly of a Zn-Binding Protein Cryptand via Templated Disulfide Bonds

Simultaneously strong and reversible through redox chemistry, disulfide bonds play a unique and often irreplaceable role in the formation of biological and synthetic assemblies. In an approach inspired by supramolecular chemistry, we report here that engineered noncovalent interactions on the surface of a monomeric protein can template its assembly into a unique cryptand-like protein complex (^C81/C96RIDC1₄) by guiding the selective formation of multiple disulfide bonds across different interfaces. Owing to its highly interconnected framework, ^C81/C96RIDC1₄ is well preorganized for metal coordination in its interior, can support a large internal cavity surrounding the metal sites, and can withstand significant alterations in inner-sphere metal coordination. ^C81/C96RIDC1₄ self-assembles with high fidelity and yield in the periplasmic space of <i>E. coli</i> cells, where it can successfully compete for Zn­(II) binding

In Vitro and Cellular Self-Assembly of a Zn-Binding Protein Cryptand via Templated Disulfide Bonds

Simultaneously strong and reversible through redox chemistry, disulfide bonds play a unique and often irreplaceable role in the formation of biological and synthetic assemblies. In an approach inspired by supramolecular chemistry, we report here that engineered noncovalent interactions on the surface of a monomeric protein can template its assembly into a unique cryptand-like protein complex (^C81/C96RIDC1₄) by guiding the selective formation of multiple disulfide bonds across different interfaces. Owing to its highly interconnected framework, ^C81/C96RIDC1₄ is well preorganized for metal coordination in its interior, can support a large internal cavity surrounding the metal sites, and can withstand significant alterations in inner-sphere metal coordination. ^C81/C96RIDC1₄ self-assembles with high fidelity and yield in the periplasmic space of <i>E. coli</i> cells, where it can successfully compete for Zn­(II) binding

In Vitro and Cellular Self-Assembly of a Zn-Binding Protein Cryptand via Templated Disulfide Bonds

Simultaneously strong and reversible through redox chemistry, disulfide bonds play a unique and often irreplaceable role in the formation of biological and synthetic assemblies. In an approach inspired by supramolecular chemistry, we report here that engineered noncovalent interactions on the surface of a monomeric protein can template its assembly into a unique cryptand-like protein complex (^C81/C96RIDC1₄) by guiding the selective formation of multiple disulfide bonds across different interfaces. Owing to its highly interconnected framework, ^C81/C96RIDC1₄ is well preorganized for metal coordination in its interior, can support a large internal cavity surrounding the metal sites, and can withstand significant alterations in inner-sphere metal coordination. ^C81/C96RIDC1₄ self-assembles with high fidelity and yield in the periplasmic space of <i>E. coli</i> cells, where it can successfully compete for Zn­(II) binding

De Novo Design of an Allosteric Metalloprotein Assembly with Strained Disulfide Bonds

A major goal in metalloprotein design is to build protein scaffolds from scratch that allow precise control over metal coordination. A particular challenge in this regard is the construction of allosteric systems in which metal coordination equilibria are coupled to other chemical events that take place elsewhere in the protein scaffold. We previously developed a metal-templated self-assembly strategy (MeTIR) to build supramolecular protein complexes with tailorable interfaces from monomeric building blocks. Here, using this strategy, we have incorporated multiple disulfide bonds into the interfaces of a Zn-templated cytochrome <i>cb</i>₅₆₂ assembly in order to create mechanical strain on the quaternary structural level. Structural and biophysical analyses indicate that this strain leads to an allosteric system in which Zn²⁺ binding and dissociation are remotely coupled to the formation and breakage of a disulfide bond over a distance of >14 Å. The breakage of this strained bond upon Zn²⁺ dissociation occurs in the absence of any reductants, apparently through a hydrolytic mechanism that generates a sulfenic acid/thiol pair

De Novo Design of an Allosteric Metalloprotein Assembly with Strained Disulfide Bonds

A major goal in metalloprotein design is to build protein scaffolds from scratch that allow precise control over metal coordination. A particular challenge in this regard is the construction of allosteric systems in which metal coordination equilibria are coupled to other chemical events that take place elsewhere in the protein scaffold. We previously developed a metal-templated self-assembly strategy (MeTIR) to build supramolecular protein complexes with tailorable interfaces from monomeric building blocks. Here, using this strategy, we have incorporated multiple disulfide bonds into the interfaces of a Zn-templated cytochrome <i>cb</i>₅₆₂ assembly in order to create mechanical strain on the quaternary structural level. Structural and biophysical analyses indicate that this strain leads to an allosteric system in which Zn²⁺ binding and dissociation are remotely coupled to the formation and breakage of a disulfide bond over a distance of >14 Å. The breakage of this strained bond upon Zn²⁺ dissociation occurs in the absence of any reductants, apparently through a hydrolytic mechanism that generates a sulfenic acid/thiol pair

Modulating Nanoparticle Superlattice Structure Using Proteins with Tunable Bond Distributions

Herein, we investigate the use of proteins with tunable DNA modification distributions to modulate nanoparticle superlattice structure. Using beta-galactosidase (βgal) as a model system, we have employed the orthogonal chemical reactivities of surface amines and thiols to synthesize protein–DNA conjugates with 36 evenly distributed or 8 specifically positioned oligonucleotides. When these are assembled into crystalline superlattices with gold nanoparticles, we find that the distribution of DNA modifications modulates the favored structure: βgal with uniformly distributed DNA bonding elements results in body-centered cubic crystals, whereas DNA functionalization of cysteines results in AB₂ packing. We probe the role of protein oligonucleotide number and conjugate size on this observation, which revealed the importance of oligonucleotide distribution in this observed assembly behavior. These results indicate that proteins with defined DNA modification patterns are powerful tools for controlling nanoparticle superlattices architecture, and establish the importance of oligonucleotide distribution in the assembly behavior of protein–DNA conjugates

Defining the Structure of a Protein–Spherical Nucleic Acid Conjugate and Its Counterionic Cloud

Protein–spherical nucleic acid conjugates (Pro-SNAs) are an emerging class of bioconjugates that have properties defined by their protein cores and dense shell of oligonucleotides. They have been used as building blocks in DNA-driven crystal engineering strategies and show promise as agents that can cross cell membranes and affect both protein and DNA-mediated processes inside cells. However, ionic environments surrounding proteins can influence their activity and conformational stability, and functionalizing proteins with DNA substantively changes the surrounding ionic environment in a nonuniform manner. Techniques typically used to determine protein structure fail to capture such irregular ionic distributions. Here, we determine the counterion radial distribution profile surrounding Pro-SNAs dispersed in RbCl with 1 nm resolution through <i>in situ</i> anomalous small-angle X-ray scattering (ASAXS) and classical density functional theory (DFT). SAXS analysis also reveals the radial extension of the DNA and the linker used to covalently attach the DNA to the protein surface. At the experimental salt concentration of 50 mM RbCl, Rb⁺ cations compensate ∼90% of the negative charge due to the DNA and linker. Above 75 mM, DFT calculations predict overcompensation of the DNA charge by Rb⁺. This study suggests a method for exploring Pro-SNA structure and function in different environments through predictions of ionic cloud densities as a function of salt concentration, DNA grafting density, and length. Overall, our study demonstrates that solution X-ray scattering combined with DFT can discern counterionic distribution and submolecular features of highly charged, complex nanoparticle constructs such as Pro-SNAs and related nucleic acid conjugate materials

Altering DNA-Programmable Colloidal Crystallization Paths by Modulating Particle Repulsion

Colloidal crystal engineering with DNA can be used to realize precise control over nanoparticle (NP) arrangement. Here, we investigate a case of DNA-based assembly where the properties of DNA as a polyelectrolyte brush are employed to alter a hybridization-driven NP crystallization pathway. Using the coassembly of DNA-conjugated proteins and spherical gold nanoparticles (AuNPs) as a model system, we explore how steric repulsion between noncomplementary, neighboring NPs due to overlapping DNA shells can influence their ligand-directed behavior. Specifically, our experimental data coupled with coarse-grained molecular dynamics (MD) simulations reveal that, by changing factors related to NP repulsion, two structurally distinct outcomes can be achieved. When steric repulsion between DNA−AuNPs is significantly greater than that between DNA–proteins, a lower packing density crystal lattice is favored over the structure that is predicted by design rules based on DNA hybridization considerations alone. This is enabled by the large difference in DNA density on AuNPs versus proteins and can be tuned by modulating the flexibility, and thus conformational entropy, of the DNA on the constituent particles. At intermediate ligand flexibility, the crystallization pathways are energetically similar, and the structural outcome can be adjusted using the density of DNA duplexes on DNA−AuNPs and by screening the Coulomb potential between them. Such lattices are shown to undergo dynamic reorganization upon changing the salt concentration. These data help elucidate the structural considerations necessary for understanding repulsive forces in DNA-mediated assembly and lay the groundwork for using them to increase architectural diversity in engineering colloidal crystals

Altering DNA-Programmable Colloidal Crystallization Paths by Modulating Particle Repulsion

Colloidal crystal engineering with DNA can be used to realize precise control over nanoparticle (NP) arrangement. Here, we investigate a case of DNA-based assembly where the properties of DNA as a polyelectrolyte brush are employed to alter a hybridization-driven NP crystallization pathway. Using the coassembly of DNA-conjugated proteins and spherical gold nanoparticles (AuNPs) as a model system, we explore how steric repulsion between noncomplementary, neighboring NPs due to overlapping DNA shells can influence their ligand-directed behavior. Specifically, our experimental data coupled with coarse-grained molecular dynamics (MD) simulations reveal that, by changing factors related to NP repulsion, two structurally distinct outcomes can be achieved. When steric repulsion between DNA−AuNPs is significantly greater than that between DNA–proteins, a lower packing density crystal lattice is favored over the structure that is predicted by design rules based on DNA hybridization considerations alone. This is enabled by the large difference in DNA density on AuNPs versus proteins and can be tuned by modulating the flexibility, and thus conformational entropy, of the DNA on the constituent particles. At intermediate ligand flexibility, the crystallization pathways are energetically similar, and the structural outcome can be adjusted using the density of DNA duplexes on DNA−AuNPs and by screening the Coulomb potential between them. Such lattices are shown to undergo dynamic reorganization upon changing the salt concentration. These data help elucidate the structural considerations necessary for understanding repulsive forces in DNA-mediated assembly and lay the groundwork for using them to increase architectural diversity in engineering colloidal crystals