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In Escherichia coli, ClpYQ (HslUV) is a two-component ATP-dependent protease in which ClpQ is the peptidase subunit and ClpY is the ATPase and the substrate-binding subunit. The ATP-dependent proteolysis is mediated by substrate recognition in the ClpYQ complex. ClpY has three domains, N, I, and C, and these domains are discrete and exhibit different binding preferences. In vivo, ClpYQ targets SulA, RcsA, RpoH, and TraJ molecules. In this study, ClpY was analyzed to identify the molecular determinants required for the binding of its natural protein substrates. Using yeast two-hybrid analysis, we showed that domain I of ClpY contains the residues responsible for recognition of its natural substrates, while domain C is necessary to engage ClpQ. Moreover, the specific residues that lie in the amino acid (aa) 137 to 150 (loop 1) and aa 175 to 209 (loop 2) double loops in domain I of ClpY were shown to be necessary for natural substrate interaction. Additionally, the two-hybrid system, together with random PCR mutagenesis, allowed the isolation of ClpY mutants that displayed a range of binding activities with SulA, including a mutant with no SulA binding trait. Subsequently, via methyl methanesulfonate tests and cpsB::lacZ assays with, e.g., SulA and RcsA as targets, we concluded that aa 175 to 209 of loop 2 are involved in the tethering of natural substrates, and it is likely that both loops, aa 137 to 150 and aa 175 to 209, of ClpY domain I may assist in the delivery of substrates into the inner core for ultimate degradation by ClpQ.

A novel and simple strategy for the synthesis of cobalt oxide decorated reduced graphene oxide (CoO-rGO) nanocomposites was developed as a high performance anode for lithium ion battery (LIB). The CoO-rGO nanocomposites were prepared by simultaneous reduction of GO and Co 2 + in the presence of NaBH 4 . Addition of 15 wt% rGO produced a narrow size distribution of ultra-fine CoO nanoparticles with nano-dimensional contact between CoO and rGO leading to an excellent electrochemical performance. The reversible capacity of CoO-rGO nanocomposites is between 1140 and 1260 mAh/g at the current density of 150 mA/g and sustained at 473 mAh/g at high current density of 2400 mA/g. In addition to the excellent rate capability of 15 wt% CoO-rGO, a reversible capacity of 690 mAh/g is achieved at 600 mA/g after 60 cycles. In addition, XPS spectra and XRD patterns of CoO-rGO after cycling clearly indicate the involvement of electrolyte into CoO nanoparticles during intercalation-deintercalation cycling, and lead to the transformation of Co species from Co 2 + to Co 3 + for the enhanced electrochemical performance at high current density.

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