While having multiple aminoacyl-tRNA synthetases implicated in Charcot-Marie-Tooth (CMT) disease suggests a common mechanism, a defect in enzymatic activity is not shared among the CMT-causing mutants. Protein misfolding is a common hypothesis underlying the development of many neurological diseases. Its process usually involves an initial reduction in protein stability and then the subsequent oligomerization and aggregation. Here, we study the structural effect of three CMT-causing mutations in tyrosyl-tRNA synthetase (TyrRS or YARS). Through various approaches, we found that the mutations do not induce changes in protein secondary structures, or shared effects on oligomerization state and stability. However, all mutations provide access to a surface masked in the wild-type enzyme, and that access correlates with protein misinteraction. With recent data on another CMT-linked tRNA synthetase, we suggest that an inherent plasticity, engendering the formation of alternative stable conformations capable of aberrant interactions, links the tRNA synthetase family to CMT.

The cytokine tumor necrosis factor-alpha (TNF-α) readily forms homotrimers at sub-nM concentrations to promote inflammation. For the treatment of inflammatory diseases with upregulated levels of TNF-α, a number of therapeutic antibodies are currently used as scavengers to reduce the active TNF-α concentration in patients. Despite their clinical success, the mode-of-action of different antibody formats with regard to a stabilization of the trimeric state is not entirely understood. Here, we use a biosensor with dynamic nanolevers to analyze the monomeric and trimeric states of TNF-α together with the binding kinetics of therapeutic biologics. The intrinsic trimer-to-monomer decay rate k = 1.7 × 10−3 s−1 could be measured directly using a microfluidic system, and antibody binding affinities were analyzed in the pM range. Trimer stabilization effects are quantified for Adalimumab, Infliximab, Etanercept, Certolizumab, Golimumab for bivalent and monovalent binding formats. Clear differences in trimer stabilization are observed, which may provide a deeper insight into the mode-of-action of TNF-α scavengers.

The engineering of high-performance enzymes for future sequencing and PCR technologies as well as the development of many anticancer drugs requires a detailed analysis of DNA/RNA synthesis processes. However, due to the complex molecular interplay involved, real-time methodologies have not been available to obtain comprehensive information on both binding parameters and enzymatic activities. Here we introduce a chip-based method to investigate polymerases and their interactions with nucleic acids, which employs an electrical actuation of DNA templates on microelectrodes. Two measurement modes track both the dynamics of the induced switching process and the DNA extension simultaneously to quantitate binding kinetics, dissociation constants and thermodynamic energies. The high sensitivity of the method reveals previously unidentified tight binding states for Taq and Pol I (KF) DNA polymerases. Furthermore, the incorporation of label-free nucleotides can be followed in real-time and changes in the DNA polymerase conformation (finger closing) during enzymatic activity are observable.