Proofreading tile sets: Error correction for algorithmic self-assembly

For robust molecular implementation of tile-based algorithmic self-assembly, methods for reducing errors must be developed. Previous studies suggested that by control of physical conditions, such as temperature and the concentration of tiles, errors (ε) can be reduced to an arbitrarily low rate - but at the cost of reduced speed (r) for the self-assembly process. For tile sets directly implementing blocked cellular automata, it was shown that r ≈ βε^2 was optimal. Here, we show that an improved construction, which we refer to as proofreading tile sets, can in principle exploit the cooperativity of tile assembly reactions to dramatically improve the scaling behavior to r ≈ βε and better. This suggests that existing DNA-based molecular tile approaches may be improved to produce macroscopic algorithmic crystals with few errors. Generalizations and limitations of the proofreading tile set construction are discussed

Hierarchical assembly is more robust than egalitarian assembly in synthetic capsids

Self-assembly of complex and functional materials remains a grand challenge in soft material science. Efficient assembly depends on a delicate balance between thermodynamic and kinetic effects, requiring fine-tuning affinities and concentrations of subunits. By contrast, we introduce an assembly paradigm that allows large error-tolerance in the subunit affinity and helps avoid kinetic traps. Our combined experimental and computational approach uses a model system of triangular subunits programmed to assemble into T=3 icosahedral capsids comprising 60 units. The experimental platform uses DNA origami to create monodisperse colloids whose 3D geometry is controlled to nanometer precision, with two distinct bonds whose affinities are controlled to kBT precision, quantified in situ by static light scattering. The computational model uses a coarse-grained representation of subunits, short-ranged potentials, and Langevin dynamics. Experimental observations and modeling reveal that when the bond affinities are unequal, two distinct hierarchical assembly pathways occur, in which the subunits first form dimers in one case, and pentamers in another. These hierarchical pathways produce complete capsids faster and are more robust against affinity variation than egalitarian pathways, in which all binding sites have equal strengths. This finding suggests that hierarchical assembly may be a general engineering principle for optimizing self-assembly of complex target structures

Srs2 Disassembles Rad51 Filaments by a Protein-Protein Interaction Triggering ATP Turnover and Dissociation of Rad51 from DNA

Rad51 is a DNA recombinase functioning in the repair of DNA double-strand breaks and the generation of genetic diversity by homologous recombination (HR). In the presence of ATP, Rad51 self-assembles into an extended polymer on single-stranded DNA to catalyze strand exchange. Inappropriate HR causes genomic instability, and it is normally prevented by remodeling enzymes that antagonize the activities of Rad51 nucleoprotein filaments. In yeast, the Srs2 helicase/translocase suppresses HR by clearing Rad51 polymers from single-stranded DNA. We have examined the mechanism of disassembly of Rad51 nucleoprotein filaments by Srs2 and find that a physical interaction between Rad51 and the C-terminal region of Srs2 triggers ATP hydrolysis within the Rad51 filament, causing Rad51 to dissociate from DNA. This allosteric mechanism explains the biological specialization of Srs2 as a DNA motor protein that antagonizes HR

Doctor of Philosophy

dissertationElectrostatics are a major driving force for many phenomena at the molecular level, where tuning polarity is vital for achieving control of assembly properties. For example, micelle formation and manipulation are governed by these forces. Negatively char

Characterization of Multiple Surface Loops in Escherichia coli Biotin Protein Ligase Functions

Multifunctional proteins utilize several strategies to interact with different partners, resulting in diverse cellular outcomes. Structural, thermodynamic and kinetic features of these proteins influence the switch between functions. The Escherichia coli biotin protein ligase, BirA, is a bifunctional protein essential for biotin homeostasis. It transfers biotin to the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase in its metabolic role or dimerizes and binds the biotin biosynthetic operator as a transcriptional repressor. Each function involves forming a protein-protein interaction, and because a single surface of BirA is used to form both interactions, the two are mutually exclusive. The BirA interaction surface contains several loops, two with highly conserved sequences, and the remaining with variable sequences. In this work the roles of four loops in facilitating BirA function were investigated. Amino acids from surface loops were replaced with alanine to obtain 18 alanine substituted variants. Homodimerization energetics measured using sedimentation equilibrium yielded an 8 kcal/mol range for variants from all loops. Steady-state and stopped-flow kinetic assays yielded 7 of 18 variants that exhibited slower rates than wild-type in biotin transfer to BCCP. The majority of alanine substituted variants are from constant loops. These results indicate that the biotin transfer reaction is mediated primarily through the constant loop and homodimerization is facilitated by all surface loops. The energetics of transcription repression complex assembly, which comprises contributions from dimerization and DNA binding, was assessed using DNaseI footprint titrations. Although variants exhibit a broad range in total assembly energetics, all dimers bind with similar affinities to DNA, implying independence between DNA binding and dimerization domains. The switch between functions was also investigated using inhibition DNaseI footprint titrations. A direct correlation between inhibition of repression complex assembly and rates of BirA-BCCP association was observed, reinforcing a kinetic mechanism for the switch between BirA functions. These studies indicate that multiple surface loops form the structural basis for bifunctionality, and BirA switches between protein-protein interactions through a kinetically controlled mechanism. Elucidation of structural and mechanistic aspects of the BirA functional switch enhances our understanding of how multifunctionality evolves and the mechanism of switching between biological functions

Investigating the Stoichiometry of RuBisCO Activase by Fluorescence Fluctuation Spectroscopy

abstract: Ribulose-1, 5-bisphosphate carboxylase oxygenase, commonly known as RuBisCO, is an enzyme involved in carbon fixation in photosynthetic organisms. The enzyme is subject to a mechanism-based deactivation during its catalytic cycle. RuBisCO activase (Rca), an ancillary enzyme belonging to the AAA+ family of the ATP-ases, rescues RuBisCO by facilitating the removal of the tightly bound sugar phosphates from the active sites of RuBisCO. In this work, we investigated the ATP/ADP dependent oligomerization equilibrium of fluorescently tagged Rca for a wide range of concentrations using fluorescence correlation spectroscopy. Results show that in the presence of ADP-Mg2+, the oligomerization state of Rca gradually changes in steps of two subunits. The most probable association model supports the dissociation constants (K_d) of ∼4, 1, 1 μM for the monomer-dimer, dimer-tetramer, and tetramer-hexamer equlibria, respectively. Rca continues to assemble at higher concentrations which are indicative of the formation of aggregates. In the presence of ATP-Mg2+, a similar stepwise assembly is observed. However, at higher concentrations (30-75 µM), the average oligomeric size remains relatively unchanged around six subunits per oligomer. This is in sharp contrast with observations in ADP-Mg2+, where a marked decrease in the diffusion coefficient of Rca was observed, consistent with the formation of aggregates. The estimated K_d values obtained from the analysis of the FCS decays were similar for the first steps of the assembly process in both ADP-Mg2+ and ATP-Mg2+. However, the formation of the hexamer from the tetramer is much more favored in ATP-Mg2+, as evidenced from 20 fold lower K_d associated with this assembly step. This suggests that the formation of a hexameric ring in the presence of ATP-Mg2+. In addition to that, Rca aggregation is largely suppressed in the presence of ATP-Mg2+, as evidenced from the 1000 fold larger K_d value for the hexamer-24 mer association step. In essence, a fluorescence-based method was developed to monitor in vitro protein oligomerization and was successfully applied with Rca. The results provide a strong hint at the active oligomeric structure of Rca, and this information will hopefully help the ongoing research on the mechanistic enzymology of Rca.Dissertation/ThesisPh.D. Chemistry 201

Understanding multidrug resistance in Gram-negative bacteria -- A study of a drug efflux pump AcrB and a periplasmic chaperone SurA

Multiple drug resistance (MDR) has been a severe issue in treatment and recovery from infection.Gram-negative bacteria intrinsically exhibit higher drug tolerance than Gram-positive microbes. In this thesis, two proteins involved in Gram-negative bacterial MDR were studied, AcrB and SurA. Resistance-nodulation-cell division pump AcrAB-TolC is the major MDR efflux system in Gram-negative bacteria and efficiently extrudes a broad range of substances from the cells. To study subtle conformational changes of AcrB in vivo, a reporter platform was designed. Cysteine pairs were introduced into different regions in the periplasmic domain of the protein, and the extents of disulfide bond formation were examined. Using this platform, an inactive mutant, AcrB∆loop, was created that existed as a well-folded monomer in vivo. Next, random mutageneses were performed on a functionally compromised mutant, AcrBP223G, to identify residues that restored the function loss. The mechanism of function restoration was examined. SurA is a periplasmic molecular chaperone for outer membrane biogenesis. Deletion of SurA decreased outer membrane density and bacterial drug resistance. The dependence of SurA function on structural flexibility and stability was examined. In addition, the effect of molecular crowding on SurA interaction with its outer membrane protein substrates was examined