Spatially addressable DNA nanostructures facilitate the self-assembly of heterogeneous elements with precisely controlled patterns. Here we organized discrete GOx/HRP enzyme pairs on specific DNA origami tiles with controlled inter-enzyme spacing and position. The distance between enzymes was systematically varied from 10 nm to 65 nm and the corresponding activities were evaluated. The study revealed two different distance dependent kinetic processes associated with the assembled enzyme pairs. Strongly enhanced activity was observed for those assemblies in which the enzymes were closely spaced, while the activity dropped dramatically for enzymes as little as 20 nm apart. Increasing the spacing further resulted in a much weaker distance dependence. Combined with diffusion modeling, the results suggest that Brownian diffusion of intermediates in solution governed the variations in activity for more distant enzyme pairs, while dimensionally-limited diffusion of intermediates across connected protein surfaces contributed to the enhancement in activity for closely spaced GOx/HRP assemblies. To further test the role of limited dimensional diffusion along protein surfaces, a noncatalytic protein bridge was inserted between GOx and HRP to connect their hydration shells. This resulted in substantially enhanced activity of the enzyme pair.

The general design of the swinging arm nanostructure complex is shown in Fig. 1a, where a two-enzyme cascade consisting of glucose-6 phosphate dehydrogenase (G6pDH) 19 and malic dehydrogenase (MDH) 20 is displayed on a DNA double-crossover (DX) tile scaffold 21 (DNA sequences for all structures used in this study are shown in Supplementary Figs. S1-S7).G6pDH catalyzes the oxidation of glucose-6-phosphate and the reduction of NAD + to NADH.Subsequently, MDH catalyzes the reduction of oxaloacetate to malic acid using the NADH produced by G6pDH. To facilitate channeling of NADH between G6pDH and MDH, an NAD + -3 functionalized poly(T) 20 oligonucleotide was attached to the DNA tile surface halfway between G6pDH and MDH (see Supplementary Figs. S8-S21 for a detailed description of conjugation, assembly and purification of the nanostructured complex). Fig. 1b shows a native polyacrylamide gel electrophoresis (PAGE) analysis of the assembled enzyme complex, together with various sub-complexes. Both the gel results and the chromatogram resulting from sizeexclusion chromatography ( Supplementary Fig. S21) demonstrate assembly of the G6pDH-NAD + -MDH swinging arm cascade with >80% yield. The assembled mixture was further purified by size exclusion chromatography for enzyme activity assays. Assembly of the complete complex was also visualized by atomic force microscopy (AFM) ( Fig. 1c and Supplementary Fig. S57 for larger view images), where the presence of the enzymes on the structure is confirmed by differences in height ("brightness") compared to the surface of the DNA tile.To first explore the parameters and understand the kinetics and mechanism of the restricted diffusion mediated by the ssDNA swinging arm, we developed a simplified model system ( Supplementary Fig. S22). In this model, a Cy3 reporter dye takes the place of NAD + on the single-stranded poly(T) 20 arm, whereas a BHQ fluorescence quencher and a Cy5 energy transfer acceptor dye replace one or both enzymes on selected probe positions surrounding the swinging arm (Fig. 2a). An oligonucleotide sequence (5'-ATA GTG AAA) was extended from the 5' end of the poly(T) 20 sequence and positioned halfway between the quencher and acceptor, allowing the arm to transiently hybridize to the probes that each bear the complementary sequence (5'-TTT CAC TAT) in analogy to the binding of NAD + /NADH to the dehydrogenases. To characterize the distance dependence of the diffusive transport and binding mediated by the swinging arm using smFRET, we chose a design in which a single Cy5-labeled capture probe was placed at one of three topologically accessible distances from the Cy3-labeled arm: 7 nm (21 base pairs), 14 nm (42 base pairs) and 21 nm (63 base pairs). As shown in Fig. 2b, the most efficient hybridization to the capture probe was observed at 7 nm, where ~94% of all swinging arms were associated with the Cy5 probe at equilibrium (leading to high FRET). As the distance increased, the equilibrium fraction of captured swinging arms decreased to ~58% at 14 nm and only ...

Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.

The initial electron transfer dynamics during photosynthesis have been studied in Rhodobacter sphaeroides reaction centers from wild type and 14 mutants in which the driving force and the kinetics of charge separation vary over a broad range. Surprisingly, the protein relaxation kinetics, as measured by tryptophan absorbance changes, are invariant in these mutants. By applying a reaction-diffusion model, we can fit the complex electron transfer kinetics of each mutant quantitatively, varying only the driving force. These results indicate that initial photosynthetic charge separation is limited by protein dynamics rather than by a static electron transfer barrier.

Mutations were made in four residues near the bacteriochlorophyll cofactors of the photosynthetic reaction center from Rhodobacter sphaeroides. These mutations, L131 Leu to His and M160 Leu to His, near the dimer bacteriochlorophylls, and M203 Gly to Asp and L177 Ile to Asp, near the monomer bacteriochlorophylls, were designed to result in the placement of a hydrogen bond donor group near the ring V keto carbonyl of each bacteriochlorophyll. Perturbations of the electronic structures of the bacteriochlorophylls in the mutants are indicated by additional resolved transitions in the bacteriochlorophyll absorption bands in steady-state low-temperature and time-resolved room temperature spectra in three of the resulting mutant reaction centers. The major effect of the two mutations near the dimer was an increase up to 80 mV in the donor oxidation-reduction midpoint potential. Correspondingly, the calculated free energy difference between the excited state of the primary donor and the initial charge separated state decreased by up to 55 mV, the initial forward electron-transfer rate was up to 4 times slower, and the rate of charge recombination between the primary quinone and the donor was approximately 30% faster in these two mutants compared to the wild type. The two mutations near the monomer bacteriochlorophylls had minor changes of 25 mV or less in the donor oxidation-reduction potential, but the mutation close to the monomer bacteriochlorophyll on the active branch resulted in a roughly 3-fold decrease in the rate of the initial electron transfer.