A fully unsupervised compartment-on-demand platform for precise nanoliter assays of time-dependent steady-state enzyme kinetics and inhibition.

The ability to miniaturize biochemical assays in water-in-oil emulsion droplets allows a massive scale-down of reaction volumes, so that high-throughput experimentation can be performed more economically and more efficiently. Generating such droplets in compartment-on-demand (COD) platforms is the basis for rapid, automated screening of chemical and biological libraries with minimal volume consumption. Herein, we describe the implementation of such a COD platform to perform high precision nanoliter assays. The coupling of a COD platform to a droplet absorbance detection set-up results in a fully automated analytical system. Michaelis-Menten parameters of 4-nitrophenyl glucopyranoside hydrolysis by sweet almond β-glucosidase can be generated based on 24 time-courses taken at different substrate concentrations with a total volume consumption of only 1.4 μL. Importantly, kinetic parameters can be derived in a fully unsupervised manner within 20 min: droplet production (5 min), initial reading of the droplet sequence (5 min), and droplet fusion to initiate the reaction and read-out over time (10 min). Similarly, the inhibition of the enzymatic reaction by conduritol B epoxide and 1-deoxynojirimycin was measured, and Ki values were determined. In both cases, the kinetic parameters obtained in droplets were identical within error to values obtained in titer plates, despite a >10(4)-fold volume reduction, from micro- to nanoliters

Ultrahigh-throughput discovery of promiscuous enzymes by picodroplet functional metagenomics

This is the final version of the article. It was first available from NPG via http://dx.doi.org/10.1038/ncomms10008Unculturable bacterial communities provide a rich source of biocatalysts, but their experimental discovery by functional metagenomics is difficult, because the odds are stacked against the experimentor. Here we demonstrate functional screening of a million-membered metagenomic library in microfluidic picolitre droplet compartments. Using bait substrates, new hydrolases for sulfate monoesters and phosphotriesters were identified, mostly based on promiscuous activities presumed not to be under selection pressure. Spanning three protein superfamilies, these break new ground in sequence space: promiscuity now connects enzymes with only distantly related sequences. Most hits could not have been predicted by sequence analysis, because the desired activities have never been ascribed to similar sequences, showing how this approach complements bioinformatic harvesting of metagenomic sequencing data. Functional screening of a library of unprecedented size with excellent assay sensitivity has been instrumental in identifying rare genes constituting catalytically versatile hubs in sequence space as potential starting points for the acquisition of new functions.This research was funded by the Engineering and Physical Sciences Research Council (EPSRC) and the Biological and Biotechnological Research Council (BBSRC). FH is an ERC Starting Investigator. PYC holds a fellowship of the EU ITN PhosChemRec, CM of the ITN ProSA and ENEFP. BK and MM were supported by postdoctoral Marie-Curie fellowships. The authors thank Sean Devenish and Nobuhiko Tokuriki for useful comments on the manuscript and Gabrielle Potocki-Veronese and Stephane Emond for help with the design of the library strategy. We thank Raindance for the gift of EA surfactant

Mouse antibody of IgM class is prone to non-enzymatic cleavage between CH1 and CH2 domains

Abstract IgM is a multivalent antibody which evolved as a first line defense of adaptive immunity. It consists of heavy and light chains assembled into a complex oligomer. In mouse serum there are two forms of IgM, a full-length and a truncated one. The latter contains μ’ chain, which lacks a variable region. Although μ’ chain was discovered many years ago, its origin has not yet been elucidated. Our results indicate that μ’ chain is generated from a full-length heavy chain by non-enzymatic cleavage of the protein backbone. The cleavage occurred specifically after Asn209 and is prevented by mutating this residue into any other amino acid. The process requires the presence of other proteins, preferentially with an acidic isoelectric point, and is facilitated by neutral or alkaline pH. This unique characteristic of the investigated phenomenon distinguishes it from other, already described, Asn-dependent protein reactions. A single IgM molecule is able to bind up to 12 epitopes via its antigen binding fragments (Fabs). The cleavage at Asn209 generates truncated IgM molecules and free Fabs, resulting in a reduced IgM valence and probably affecting IgM functionality in vivo

Catalytic Mechanism of Bacteriophage T4 Rad50 ATP Hydrolysis

Spontaneous double-strand breaks (DSBs) are one of the most deleterious forms of DNA damage, and their improper repair can lead to cellular dysfunction. The Mre11 and Rad50 proteins, a nuclease and an ATPase, respectively, form a well-conserved complex that is involved in the initial processing of DSBs. Here we examine the kinetic and catalytic mechanism of ATP hydrolysis by T4 Rad50 (gp46) in the presence and absence of Mre11 (gp47) and DNA. Single-turnover and pre-steady state kinetics on the wild-type protein indicate that the rate-limiting step for Rad50, the MR complex, and the MR-DNA complex is either chemistry or a conformational change prior to catalysis. Pre-steady state product release kinetics, coupled with viscosity steady state kinetics, also supports that the binding of DNA to the MR complex does not alter the rate-limiting step. The lack of a positive deuterium solvent isotope effect for the wild type and several active site mutants, combined with pH–rate profiles, implies that chemistry is rate-limiting and the ATPase mechanism proceeds via an asymmetric, dissociative-like transition state. Mutation of the Walker A/B and H-loop residues also affects the allosteric communication between Rad50 active sites, suggesting possible routes for cooperativity between the ATP active sites

The evolution of multiple active site configurations in a designed enzyme

Developments in computational chemistry, bioinformatics, and laboratory evolution have facilitated the de novo design and catalytic optimization of enzymes. Besides creating useful catalysts, the generation and iterative improvement of designed enzymes can provide valuable insight into the interplay between the many phenomena that have been suggested to contribute to catalysis. In this work, we follow changes in conformational sampling, electrostatic preorganization, and quantum tunneling along the evolutionary trajectory of a designed Kemp eliminase. We observe that in the Kemp Eliminase KE07, instability of the designed active site leads to the emergence of two additional active site configurations. Evolutionary conformational selection then gradually stabilizes the most efficient configuration, leading to an improved enzyme. This work exemplifies the link between conformational plasticity and evolvability and demonstrates that residues remote from the active sites of enzymes play crucial roles in controlling and shaping the active site for efficient catalysis

First Fit Ergebnisse nach Bonebridge Implantation bei Patienten mit Gehörgangsatresie

Einleitung: Seit 2012 ist das teilimplantierbare Hörsystem Bonebridge der Fa. Med-El für Schalleitungs-, kombinierte Schwerhörigkeiten oder einseitige Taubheit verfügbar. Ein besonderes Patientenkollektiv stellen Patienten mit Gehörgangsatresie dar. Wir haben retrospektiv die audiometrischen Daten von 17 Atresie Patienten (4 bilateral) im Alter von 3-48 Jahren ± 13 vor der Implantation mit den vorliegenden First Fit Ergebnissen nach der Implantation verglichen. Ergebnisse: Unversorgt fand sich eine mittlere Luftleitungs-Hörschwelle von 63,5 dBHL ± 12,2 dB HL mit einer Schalleitungskomponente von 49,1 dBHL ± 18 dBHL sowie 4,58 dB S/N ± 7,67 im OLKISA. Die Implantation des Bonebridge Systems erbrachte sowohl eine signifikante Verbesserung der mittleren Luftleitungs-Hörschwelle 32,25 dB ± 12 dBHL, Reduzierung der mittleren Schalleitungskomponente 17,8 dB ± 15,9 dBHL und Verbesserung des S/N um 6,8 dB ± 1,1 im OLKISA. Schlussfolgerung: Bereits audiologische Erst-Anpassungsergebnisse zeigen eine signifikante Hörverbesserung. Mit einem weiteren Zugewinn ist durch Hörgewöhnung zu rechnen.Der Erstautor gibt keinen Interessenkonflikt an

USER friendly DNA recombination (USERec): a simple and flexible near homology-independent method for gene library construction.

USER friendly DNA recombination (USERec) is introduced as a near homology-independent method that allows the simultaneous recombination of an unprecedented number of 10 DNA fragments (approximately 40-400 bp) within a day. The large number of fragments and their ease of preparation enables the creation of libraries of much larger genetic diversity (potentially approximately 10(10)-10(11) sequences) than current alternative methods based on DNA truncation (ITCHY, SCRATCHY and SHIPREC) or type IIb restriction enzymes (SISDC). At the same time, the frequency of frameshifts in the recombined library is low (90% of the recombined sequences are in frame). Compared to overlap extension PCR, USERec also requires much reduced crossover sequence constraints (only a 5'-AN(4-8)T-3' motif) and fewer experimental steps. Based on its simplicity and flexibility, and the accessibility of large and high quality recombined DNA libraries, USERec is established as a convenient alternative for the combinatorial assembly of gene fragments (e.g. exon or domain shuffling) and for a number of applications in gene library construction, such as loop grafting and multi-site-directed or random mutagenesis