High electric fields at the edge of the substrate metallization can give rise to partial discharge within power electronic modules and can lead to eventual failure. This paper examines the use of silicone gels filled with barium titanate to reduce the electric field enhancement at the edge of substrate metallization and therefore increase partial discharge inception voltages. The barium titanate filled gel produces a dielectric in which the relative permittivity is increased over a plain gel and that also exhibits a dependence on electric field. The theoretical electric field reduction that can be achieved in a power electronic module through the use of filled gels is demonstrated and compared against experimental measurements including the trial of the technique in some commercial modules. As promising results are achieved, consideration is also given to the effect of the barium titanate filler on the viscosity of the gel and the thermal conductivity, two key manufacturing issues.

Edited by Peter CresswellProteins are often engineered to have higher affinity for their ligands to achieve therapeutic benefit. For example, many studies have used phage or yeast display libraries of mutants within complementarity-determining regions to affinity mature antibodies and T cell receptors (TCRs). However, these approaches do not allow rapid assessment or evolution across the entire interface. By combining directed evolution with deep sequencing, it is now possible to generate sequence fitness landscapes that survey the impact of every amino acid substitution across the entire protein-protein interface. Here we used the results of deep mutational scans of a TCR-peptide-MHC interaction to guide mutational strategies. The approach yielded stable TCRs with affinity increases of >200-fold. The substitutions with the greatest enrichments based on the deep sequencing were validated to have higher affinity and could be combined to yield additional improvements. We also conducted in silico binding analyses for every substitution to compare them with the fitness landscape. Computational modeling did not effectively predict the impacts of mutations distal to the interface and did not account for yeast display results that depended on combinations of affinity and protein stability. However, computation accurately predicted affinity changes for mutations within or near the interface, highlighting the complementary strengths of computational modeling and yeast surface display coupled with deep mutational scanning for engineering high affinity TCRs.The process of increasing the affinity of a protein occurs naturally with antibodies, where somatic mutation within the variable region genes is followed by antigen-driven selection of B cells that express membrane-bound antibodies. In contrast, T cell receptors (TCRs) 3 do not undergo somatic mutations and bind to their antigen, a peptide-MHC (pepMHC), with low (micromolar) affinities. However, improvements in TCR affinity to the same levels of antibodies can be achieved by in vitro approaches involving the generation of mutant TCR libraries followed by antigen selection (1-3).For therapeutic purposes, the affinity of a variety of proteinprotein interactions, and especially antibody-antigen interactions, has been enhanced using in vitro directed evolution approaches, including phage, yeast, ribosomal, and mammalian display (e.g. see Refs. 4 -7). These methods rely on the generation of large libraries of mutants at residues within the proteinprotein interface, followed by several rounds of selection for desired parameters (such as affinity, stability, and expression levels) (8, 9).Although directed evolution using larger degenerate libraries has been successful, the most recent techniques involving deep sequencing of single-codon libraries have the potential both to provide mechanistic structural information about a binding site and at the same time to provide leads for affinity improvements. Sequence fitness landscapes have successfully been utilized to map protein-DNA...

Cellular uptake of platinum-based antitumor drugs is a critical step in the mechanism of the drug action and associated resistance, and deeper understanding of this step may inspire development of novel methods for new drugs with reduced resistance. Human copper transporter 1 (hCtr1), a copper influx protein, was recently found to facilitate the cellular entry of several platinum drugs. In the work reported here, we constructed a Met- and His-rich 20mer peptide (hCtr1-N20) corresponding to the N-terminal domain of hCtr1, which is the essential domain of hCtr1 for transporting platinum drugs. The interactions of the peptide with cisplatin and its analogues, including transplatin, carboplatin, oxaliplatin, and [Pt(L: -Met)Cl(2)], were explored at the molecular level. Electrospray ionization (ESI) mass spectrometry (MS) data revealed that all of the platinum(II) complexes used in present study can bind to hCtr1-N20 in 1:1 and 2:1 stoichiometry. Four Met residues should be involved in binding to cis-platinum complexes on the basis of the tandem MS spectrometry and previously reported data. Time-dependent 2D [(1)H,(15)N] heteronuclear single quantum coherence NMR spectra indicate the reaction of cisplatin with hCtr1-N20 is a stepwise process. The intermediate, however, is transient, which is consistent with the ESI-MS results. Time-dependent ESI-MS data revealed that the geometry and the properties of both the leaving and the nonleaving groups of platinum(II) complexes play essential roles in controlling the reactivity and formation of the final products with hCtr1-N20.

To address the role of the secondary hydroxyl group of heme a/o in heme-copper oxidases, we incorporated Fe(III)-2,4 (4,2) hydroxyethyl vinyl deuterioporphyrin IX, as a heme o mimic, into the engineered heme-copper center in myoglobin (sperm whale myoglobin L29H/F43H, called Cu(B)Mb). The only difference between the heme b of myoglobin and the heme o mimic is the substitution of one of the vinyl side chains of the former with a hydroxyethyl group of the latter. This substitution resulted in an approximately 4 nm blue shift in the Soret band and approximately 20 mV decrease in the heme reduction potential. In a control experiment, the heme b in Cu(B)Mb was also replaced with a mesoheme, which resulted in an approximately 13 nm blue shift and approximately 30 mV decrease in the heme reduction potential. Kinetic studies of the heme o mimic-substituted Cu(B)Mb showed significantly different reactivity toward copper-dependent oxygen reduction from that of the b-type Cu(B)Mb. In reaction with O2, Cu(B)Mb with a native heme b showed heme oxygenase activity by generating verdoheme in the presence of Cu(I). This heme degradation reaction was slowed by approximately 19-fold in the heme o mimic-substituted Cu(B)Mb (from 0.028 s(-1) to 0.0015 s(-1)), while the mesoheme-substituted Cu(B)Mb shared a similar heme degradation rate with that of Cu(B)Mb (0.023 s(-1)). No correlation was found between the heme reduction potential and its O2 reactivity. These results strongly suggest the critical role of the hydroxyl group of heme o in modulating heme-copper oxidase activity through participation in an extra hydrogen-bonding network.

Advanced spectroscopic techniques coupled with DFT calculations reveal the vibrational dynamics of the iron in stable dioxygen complexes with myoglobin and with a mutant engineered to model the catalytic site of heme-copper oxidases. The unprecedented level of detail will constrain computational modelling of reactions with oxygen.