Droplets as reaction compartments for protein nanotechnology

Extreme miniaturization of biological and chemical reactions in pico- to nanoliter microdroplets is emerging as an experimental paradigm that enables more experiments to be carried out with much lower sample consumption, paving the way for high-throughput experiments. This review provides the protein scientist with an experimental framework for (a) formation of polydisperse droplets by emulsification or, alternatively, of monodisperse droplets using microfluidic devices; (b) construction of experimental rigs and microfluidic chips for this purpose; and (c) handling and analysis of droplets

Conserved main-chain peptide distortions: A proposed role for Ile203 in catalysis by dihydrodipicolinate synthase

In recent years, dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) has received considerable attention from a mechanistic and structural viewpoint. DHDPS catalyzes the reaction of (S)-aspartate-β-semialdehyde with pyruvate, which is bound via a Schiff base to a conserved active-site lysine (Lys161 in the enzyme from Escherichia coli). To probe the mechanism of DHDPS, we have studied the inhibition of E. coli DHDPS by the substrate analog, β-hydroxypyruvate. The K i was determined to be 0.21 (±0.02) mM, similar to that of the allosteric inhibitor, (S)-lysine, and β-hydroxypyruvate was observed to cause time-dependent inhibition. The inhibitory reaction with β-hydroxypyruvate could be qualitatively followed by mass spectrometry, which showed initial noncovalent adduct formation, followed by the slow formation of the covalent adduct. It is unclear whether β-hydroxypyruvate plays a role in regulating the biosynthesis of meso-diaminopimelate and (S)-lysine in E. coli, although we note that it is present in vivo. The crystal structure of DHDPS complexed with β-hydroxypyruvate was solved. The active site clearly showed the presence of the inhibitor covalently bound to the Lys161. Interestingly, the hydroxyl group of β-hydroxypyruvate was hydrogen-bonded to the main-chain carbonyl of Ile203. This provides insight into the possible catalytic role played by this peptide unit, which has a highly strained torsion angle (ω ∼201°). A survey of the known DHDPS structures from other organisms shows this distortion to be a highly conserved feature of the DHDPS active site, and we propose that this peptide unit plays a critical role in catalysis

Kinetic fingerprints differentiate the mechanisms of action of anti-Aβ antibodies

The amyloid cascade hypothesis, according to which the self-assembly of amyloid-β peptide (Aβ) is a causative process in Alzheimer’s disease, has driven many therapeutic efforts for the past 20 years. Failures of clinical trials investigating Aβ-targeted therapies have been interpreted as evidence against this hypothesis, irrespective of the characteristics and mechanisms of action of the therapeutic agents, which are highly challenging to assess. Here, we combine kinetic analyses with quantitative binding measurements to address the mechanism of action of four clinical stage anti-Aβ antibodies, aducanumab, gantenerumab, bapineuzumab and solanezumab. We quantify the influence of these antibodies on the aggregation kinetics and on the production of oligomeric aggregates and link these effects to the affinity and stoichiometry of each antibody for monomeric and fibrillar forms of Aβ. Our results reveal that, uniquely among these four antibodies, aducanumab dramatically reduces the flux of Aβ oligomers

Plant DHDPR forms a dimer with unique secondary structure features that preclude higher-order assembly

Dihydrodipicolinate reductase (DHDPR) catalyses the second reaction in the diaminopimelate pathway of lysine biosynthesis in bacteria and plants. In contrast with the tetrameric bacterial DHDPR enzymes, we show that DHDPR from Vitis vinifera (grape) and Selaginella moellendorffii are dimeric in solution. In the present study, we have also determined the crystal structures of DHDPR enzymes from the plants Arabidopsis thaliana and S. moellendorffii, which are the first dimeric DHDPR structures. The analysis of these models demonstrates that the dimer forms through the intra-strand interface, and that unique secondary features in the plant enzymes block tetramer assembly. In addition, we have also solved the structure of tetrameric DHDPR from the pathogenic bacteria Neisseria meningitidis. Measuring the activity of plant DHDPR enzymes showed that they are much more prone to substrate inhibition than the bacterial enzymes, which appears to be a consequence of increased flexibility of the substrate-binding loop and higher affinity for the nucleotide substrate. This higher propensity to substrate inhibition may have consequences for ongoing efforts to increase lysine biosynthesis in plants