Engineered plyab nanopores and uses thereof.

The invention relates generally to the field of nanopores and the use thereof in analyzing biopolymers. In particular, it relates to engineered biological nanopores and their application in single molecule analysis, such as single molecule protein identification

Soluble oligomerization provides a beneficial fitness effect on destabilizing mutations

Mutations create the genetic diversity on which selective pressures can act, yet also create structural instability in proteins. How, then, is it possible for organisms to ameliorate mutation-induced perturbations of protein stability while maintaining biological fitness and gaining a selective advantage? Here we used a new technique of site-specific chromosomal mutagenesis to introduce a selected set of mostly destabilizing mutations into folA - an essential chromosomal gene of E. coli encoding dihydrofolate reductase (DHFR) - to determine how changes in protein stability, activity and abundance affect fitness. In total, 27 E.coli strains carrying mutant DHFR were created. We found no significant correlation between protein stability and its catalytic activity nor between catalytic activity and fitness in a limited range of variation of catalytic activity observed in mutants. The stability of these mutants is strongly correlated with their intracellular abundance; suggesting that protein homeostatic machinery plays an active role in maintaining intracellular concentrations of proteins. Fitness also shows a significant correlation with intracellular abundance of soluble DHFR in cells growing at 30oC. At 42oC, on the other hand, the picture was mixed, yet remarkable: a few strains carrying mutant DHFR proteins aggregated rendering them nonviable, but, intriguingly, the majority exhibited fitness higher than wild type. We found that mutational destabilization of DHFR proteins in E. coli is counterbalanced at 42oC by their soluble oligomerization, thereby restoring structural stability and protecting against aggregation

Electro-osmotic capture and ionic discrimination of peptide and protein biomarkers with FraC nanopores

Biological nanopores are nanoscale sensors employed for high-throughput, low-cost, and long read-length DNA sequencing applications. The analysis and sequencing of proteins, however, is complicated by their folded structure and non-uniform charge. Here we show that an electro-osmotic flow through Fragaceatoxin C (FraC) nanopores can be engineered to allow the entry of polypeptides at a fixed potential regardless of the charge composition of the polypeptide. We further use the nanopore currents to discriminate peptide and protein biomarkers from 25 kDa down to 1.3 kDa including polypeptides differing by one amino acid. On the road to nanopore proteomics, our findings represent a rationale for amino-acid analysis of folded and unfolded polypeptides with nanopores.Biological nanopore-based protein sequencing and recognition is challenging due to the folded structure or non-uniform charge of peptides. Here the authors show that engineered FraC nanopores can overcome these problems and recognize biomarkers in the form of oligopeptides, polypeptides and folded proteins

Direct electrical quantification of glucose and asparagine from bodily fluids using nanopores

Crucial steps in the miniaturisation of biosensors are the conversion of a biological signal into an electrical current as well as the direct sampling of bodily fluids. Here we show that protein sensors in combination with a nanopore, acting as an electrical transducer, can accurately quantify metabolites in real time directly from nanoliter amounts of blood and other bodily fluids. Incorporation of the nanopore into portable electronic devices will allow developing sensitive, continuous, and non-invasive sensors for metabolites for point-of-care and home diagnostics

A biophysical protein folding model accounts for most mutational fitness effects in viruses

Fitness effects of mutations fall on a continuum ranging from lethal to deleterious to beneficial. The distribution of fitness effects (DFE) among random mutations is an essential component of every evolutionary model and a mathematical portrait of robustness. Recent experiments on five viral species all revealed a characteristic bimodal shaped DFE, featuring peaks at neutrality and lethality. However, the phenotypic causes underlying observed fitness effects are still unknown, and presumably thought to vary unpredictably from one mutation to another. By combining population genetics simulations with a simple biophysical protein folding model, we show that protein thermodynamic stability accounts for a large fraction of observed mutational effects. We assume that moderately destabilizing mutations inflict a fitness penalty proportional to the reduction in folded protein, which depends continuously on folding free energy (\Delta G). Most mutations in our model affect fitness by altering \Delta G, while, based on simple estimates, \approx10% abolish activity and are unconditionally lethal. Mutations pushing \Delta G>0 are also considered lethal. Contrary to neutral network theory, we find that, in mutation/selection/drift steady-state, high mutation rates (m) lead to less stable proteins and a more dispersed DFE, i.e. less mutational robustness. Small population size (N) also decreases stability and robustness. In our model, a continuum of non-lethal mutations reduces fitness by \approx2% on average, while \approx10-35% of mutations are lethal, depending on N and m. Compensatory mutations are common in small populations with high mutation rates. More broadly, we conclude that interplay between biophysical and population genetic forces shapes the DFE.Comment: Main text: 12 pages, 5 figures Supplementary Information: 10 pages, 5 figure

Regulatory network structure determines patterns of intermolecular epistasis

Most phenotypes are determined by molecular systems composed of specifically interacting molecules. However, unlike for individual components, little is known about the distributions of mutational effects of molecular systems as a whole. We ask how the distribution of mutational effects of a transcriptional regulatory system differs from the distributions of its components, by first independently, and then simultaneously, mutating a transcription factor and the associated promoter it represses. We find that the system distribution exhibits increased phenotypic variation compared to individual component distributions - an effect arising from intermolecular epistasis between the transcription factor and its DNA-binding site. In large part, this epistasis can be qualitatively attributed to the structure of the transcriptional regulatory system and could therefore be a common feature in prokaryotes. Counter-intuitively, intermolecular epistasis can alleviate the constraints of individual components, thereby increasing phenotypic variation that selection could act on and facilitating adaptive evolution

Mutations Closer to the Active Site Improve the Promiscuous Aldolase Activity of 4-Oxalocrotonate Tautomerase More Effectively than Distant Mutations

The enzyme 4-oxalocrotonate tautomerase (4-OT), which catalyzes enol-keto tautomerization as part of a degradative pathway for aromatic hydrocarbons, promiscuously catalyzes various carbon-carbon bond-forming reactions. These include the aldol condensation of acetaldehyde with benzaldehyde to yield cinnamaldehyde. Here, we demonstrate that 4-OT can be engineered into a more efficient aldolase for this condensation reaction, with a &gt;5000-fold improvement in catalytic efficiency (kcat /Km ) and a &gt;10(7) -fold change in reaction specificity, by exploring small libraries in which only "hotspots" are varied. The hotspots were identified by systematic mutagenesis (covering each residue), followed by a screen for single mutations that give a strong improvement in the desired aldolase activity. All beneficial mutations were near the active site of 4-OT, thus underpinning the notion that new catalytic activities of a promiscuous enzyme are more effectively enhanced by mutations close to the active site.</p