Sierin Lim

Graphene/carbon cloth anode for high-performance mediatorless microbial fuel cells.

Graphene was electrochemically deposited on carbon cloth to fabricate an anode for a Pseudomonas aeruginosa mediatorless microbial fuel cell (MFC). The graphene modification improved power density and energy conversion efficiency by 2.7 and 3 times, respectively. The improvement is attributed to the high biocompatibility of graphene which promotes bacteria growth on the electrode surface that results in the creation of more direct electron transfer activation centers and stimulates excretion of mediating molecules for higher electron transfer rate. A parallel bioelectrocatalytic mechanism consisting of simultaneous direct electron transfer and cell-excreted mediator-enabled electron transfer was established in the P. aeruginosa-catalyzed MFC. This study does not only offer fundamental insights into MFC reactions, but also suggests a low cost manufacturing process to fabricate high power MFCs for practical applications.

Protein-Based Memristive Nanodevices

6 Memristors (memory resistors), represent the fourth fundamental two-terminal circuit element following the resistor, the capacitor, and the inductor. [ 1 , 2 ] Memristors have attracted much attention owing to their potential application in nanoelectronic memories, computer logic, neuromorphic computer architectures, and so on. [ 3–6 ] Williams et al. were the fi rst to demonstrate a solid-state memristive device based on a titanium dioxide thin fi lm, which was inserted between two Pt electrodes. [ 2 ] Thus far, various materials such as metal oxides, [ 7–10 ] chalcogenides, [ 11 ] amorphous silicon, [ 12–15 ] or carbon, [ 16 ] as well as polymer–nanoparticle composite materials [ 17 ] have been demonstrated to exhibit memristive phenomena. Many activities in life exhibit memory behavior. Substantial research has focused on biomolecules serving as computing elements, [ 18–20 ] and as such natural biomaterials may have the potential to be exploited as electronic memristors. Some types of proteins, especially redox proteins, have been shown to act as electronic materials when inserted between electrodes to achieve solid-state electron-transport junctions. [ 21–23 ] In this communication, protein-based bipolar memristive nanodevices in which proteins are embedded into the nanogaps are demonstrated. Several methods, such as mechanical break-junction techniques, [ 24 , 25 ] electromigration, [ 26–28 ] and shadow mask evaporation [ 29–31 ] have all been used to fabricate nanogaps; nanostructures that have metal-electrode pairs separated by a few nanometers that enable an electrical contact to target molecules. On-wire lithography (OWL), a chemistry-based nanofabrication technique, is a high-throughput method used to prepare nanogaps, affording control of feature sizes down to typical molecular dimensions. [ 32 , 33 ] This method relies on the template-directed synthesis of nanowires in an anodized aluminum oxide membrane by the electrochemical deposition of desired metal materials. The gap size is controlled by the thickness of a thin sacrifi cial layer of Ni,

Thermostability and molecular encapsulation within an engineered caged protein scaffold

Self‐assembling biological complexes such as viral capsids have been manipulated to function in innovative nanotechnology applications. The E2 component of pyruvate dehydrogenase from Bacillus stearothermophilus forms a dodecahedral complex and potentially provides another platform for these purposes. In this investigation, we show that this protein assembly exhibits unusual stability and can be modified to encapsulate model drug molecules. To distill the E2 protein down to its structural scaffold core, we synthesized a truncated gene optimized for expression in Escherichia coli. The correct assembly and dodecahedral structure of the resulting scaffold was confirmed with dynamic light scattering and transmission electron microscopy. Using circular dichroism and differential scanning calorimetry, we found the thermostability of the complex to be unusually high, with an onset temperature of unfolding at 81.1 ± 0.9°C and an apparent midpoint unfolding temperature of 91.4 ± 1.4°C. To evaluate the potential of this scaffold for encapsulation of guest molecules, we made variants at residues 381 and 239 which altered the physicochemical properties of the hollow internal cavity. These mutants, yielding 60 and 120 mutations within this cavity, assembled into the correct architecture and exhibited high thermostability that was comparable to the wild‐type scaffold. To show the applicability of this scaffold, two different fluorescent dye molecules were covalently coupled to the cysteine mutant at site 381. We demonstrate that these mutations can introduce non‐native functionality and enable molecular encapsulation within the cavity while still retaining the dodecahedral structure. The unusually robust nature of this scaffold and its amenability to internal changes reveal its potential for nanoscale applications. Biotechnol. Bioeng. 2008;101: 654–664.

Design of a pH-dependent molecular switch in a caged protein platform.

Self-assembling protein cages provide a wide range of possible applications in nanotechnology. We report the first example of an engineered pH-dependent molecular switch in a virus-like particle. By genetically manipulating the subunit-subunit interface of the E2 subunit of pyruvate dehydrogenase, we introduce pH-responsive assembly into a scaffold that is natively stable at both pH 5.0 and 7.4. The redesigned protein module yields an intact, stable particle at pH 7.4 that dissociates at pH 5.0. This triggered behavior is especially relevant for applications in therapeutic delivery.

Iron-based ferritin nanocore as a contrast agent

Self-assembling protein cages have been exploited as templates for nanoparticle synthesis. The ferritin molecule, a protein cage present in most living systems, stores excess soluble ferrous iron in the form of an insoluble ferric complex within its cavity. Magnetic nanocores formed by loading excess iron within an engineered ferritin from Archaeoglobus fulgidus (AfFtn-AA) were studied as a potential magnetic resonance (MR) imaging contrast agent. The self-assembly characteristics of the AfFtn-AA were investigated using dynamic light scattering technique and size exclusion chromatography. Homogeneous size distribution of the assembled nanoparticles was observed using transmission electron microscopy. The magnetic properties of iron-loaded AfFtn-AA were studied using vibrating sample magnetometry. Images obtained from a 3.0 T whole-body MRI scanner showed significant brightening of T1 images and signal loss of T2 images with increased concentrations of iron-loaded AfFtn-AA. The analysis of the MR image intensities showed extremely high R2 values (5300 mM−1 s−1) for the iron-loaded AfFtn-AA confirming its potential as a T2 contrast agent.

A Novel Archaeal Alanine Dehydrogenase Homologous to Ornithine Cyclodeaminase and μ-Crystallin

A novel alanine dehydrogenase (AlaDH) showing no significant amino acid sequence homology with previously known bacterial AlaDHs was purified to homogeneity from the soluble fraction of the hyperthermophilic archaeon Archaeoglobus fulgidus. AlaDH catalyzed the reversible, NAD+-dependent deamination of L-alanine to pyruvate and NH4+. NADP(H) did not serve as a coenzyme. The enzyme is a homodimer of 35 kDa per subunit. The Km values for L-alanine, NAD+, pyruvate, NADH, and NH4+ were estimated at 0.71, 0.60, 0.16, 0.02, and 17.3 mM, respectively. The A. fulgidus enzyme exhibited its highest activity at about 82 degrees C (203 U/mg for reductive amination of pyruvate) yet still retained 30% of its maximum activity at 25 degrees C. The thermostability of A. fulgidus AlaDH was increased by more than 10-fold by 1.5 M KCl to a half-life of 55 h at 90 degrees C. At 25 degrees C in the presence of this salt solution, the enzyme was approximately 100% stable for more than 3 months. Closely related A. fulgidus AlaDH homologues were found in other archaea. On the basis of its amino acid sequence, A. fulgidus AlaDH is a member of the ornithine cyclodeaminase-mu-crystallin family of enzymes. Similar to the mu-crystallins, A. fulgidus AlaDH did not exhibit any ornithine cyclodeaminase activity. The recombinant human mu-crystallin was assayed for AlaDH activity, but no activity was detected. The novel A. fulgidus gene encoding AlaDH, AF1665, is designated ala.

Bioengineered Tunable Memristor Based on Protein Nanocage

Bioengineered protein-based nanodevices with tunable and reproducible memristive performance are fabricated by combining the unique high loading capacity of Archaeoglobus fulgidus ferritin with OWL-generated nanogaps. By tuning the iron amount inside ferritin, the ON/OFF ratio of conductance switching can be modulated accordingly. Higher molecular loading exhibits better memristive performance owing to the higher electrochemical activity of the ferric complex core.

pH-triggered disassembly in a caged protein complex.

Self-assembling protein cage structures have many potential applications in nanotechnology, one of which is therapeutic delivery. For intracellular targeting, pH-controlled disassembly of virus-like particles and release of their molecular cargo is particularly strategic. We investigated the potential of using histidines for introducing pH-dependent disassembly in the E2 subunit of pyruvate dehydrogenase. Two subunit interfaces likely to disrupt stability, an intratrimer interface (the N-terminus) and an intertrimer interface (methionine-425), were redesigned. Our results show that changing the identity of the putative anchor site 425 to histidine does not decrease stability. In contrast, engineering non-native pH-dependent behavior and modulating the transition pH at which disassembly occurs can be accomplished by mutagenesis of the N-terminus and by ionic strength changes. The observed pH-triggered disassembly is due to electrostatic repulsions generated by histidine protonation. These results suggest that altering the degree of electrostatic repulsion at subunit interfaces could be a generally applicable strategy for designing pH-triggered assembly in protein macromolecular structures.

Long-Range Tunneling Processes across Ferritin-Based Junctions

The mechanism of long-range charge transport across tunneling junctions with monolayers of ferritin is investigated. It is shown that the mechanism can be switched between coherent tunneling, sequential tunneling, and hopping by changing the iron content inside the ferritin. This study shows that ferritins are an interesting class of biomolecules to control charge transport.

The role of nonconserved residues of Archaeoglobus fulgidus ferritin on its unique structure and biophysical properties.

Background: Archaeoglobus fulgidus ferritin (AfFtn) assembles with unique tetrahedral symmetry and four large pores. Results: The AfFtn K150A/R151A double mutant forms a closed octahedral assembly with reduced iron release rates relative to the tetrahedral assembly. Conclusion: The K150A/R151A substitution alters the symmetry type of the ferritin cage. Significance: The AfFtn can be modulated for tuning molecular release from the cavity. Archaeoglobus fulgidus ferritin (AfFtn) is the only tetracosameric ferritin known to form a tetrahedral cage, a structure that remains unique in structural biology. As a result of the tetrahedral (2-3) symmetry, four openings (∼45 Å in diameter) are formed in the cage. This open tetrahedral assembly contradicts the paradigm of a typical ferritin cage: a closed assembly having octahedral (4-3-2) symmetry. To investigate the molecular mechanism affecting this atypical assembly, amino acid residues Lys-150 and Arg-151 were replaced by alanine. The data presented here shed light on the role that these residues play in shaping the unique structural features and biophysical properties of the AfFtn. The x-ray crystal structure of the K150A/R151A mutant, solved at 2.1 Å resolution, indicates that replacement of these key residues flips a “symmetry switch.” The engineered molecule no longer assembles with tetrahedral symmetry but forms a typical closed octahedral ferritin cage. Small angle x-ray scattering reveals that the overall shape and size of AfFtn and AfFtn-AA in solution are consistent with those observed in their respective crystal structures. Iron binding and release kinetics of the AfFtn and AfFtn-AA were investigated to assess the contribution of cage openings to the kinetics of iron oxidation, mineralization, or reductive iron release. Identical iron binding kinetics for AfFtn and AfFtn-AA suggest that Fe2+ ions do not utilize the triangular pores for access to the catalytic site. In contrast, relatively slow reductive iron release was observed for the closed AfFtn-AA, demonstrating involvement of the large pores in the pathway for iron release.