Science

Ec DOS, a heme-regulated phosphodiesterase from Escherichia coli, is composed of an N-terminal heme-bound PAS domain and a C-terminal phosphodiesterase domain. The heme redox state in the PAS domain regulates Ec DOS phosphodiesterase activity. Interestingly, the isolated heme-bound PAS fragment enhances phosphodiesterase activity of full-length Ec DOS. The enhancement is also regulated by the heme redox state of the isolated PAS domain. In the present study, we used a newly developed protein microarray system to examine the relationship between catalytic activity and the interaction of full-length Ec DOS and the isolated PAS fragment. Adenosine 3′,5′-cyclic monophosphate (cAMP), a substrate of the Ec DOS phosphodiesterase, was found to be indispensable for the interaction between Ec DOS and the PAS fragment, and two phosphodiesterase inhibitors, 3-isobutyl-methyl-xanthine and etazolate hydrochloride, hindered the interaction. In addition, an enzyme with a mutation in the putative cAMP-binding sites (H590 and H594) was unable to interact with Ec DOS and lacked enzymatic activity. These results strongly suggest a close relationship between Ec DOS phosphodiesterase activity and interaction with the isolated PAS fragment. Therefore, this study provides insights into the mechanism of how the isolated PAS domain activates Ec DOS, which has important implications for the general role of the isolated PAS domain in cells. Moreover, we found that multiple microscale analyses using the protein microarray system had several advantages over conventional affinity column methods, including the quantity of protein needed, the sensitivity, the variability of immobilized protein, and the time required for the experiment.

Biochemistry Z 0250 Structure-Function Relationships of EcDOS, a Heme-Regulated Phosphodiesterase from Escherichia coli -[65 refs.]. -(SASAKURA*, Y.; YOSHIMURA-SUZUKI, T.; KUROKAWA, H.; SHIMIZU, T.; Acc. Chem. Res. 39 (2006) 1, 37-43; Inst. Multidisc. Res. Adv. Mater., Tohoku Univ., Aoba, Sendai 980, Japan; Eng.) -Lindner 19-265

A highly sensitive microarray system for detecting proteinprotein interactions has been developed. This method was successfully applied to analyze the interactions of hemeregulated phosphodiesterase from Escherichia coli (Ec DOS). To immobilize (His) 6 -Tag fused Ec DOS, anti-(His) 6 -Tag monoclonal antibody (anti-(His) 6 -Tag mAb) was initially immobilized on the solid surface, and (His) 6 -Tag fused Ec DOS was fixed by antigen-antibody interactions. For this experiment, ProteoChip, generally suitable for antibody immobilization, was used as solid substrate. In this report, we confirm the antibody immobilization ability of ProteoChip and specific binding to the F (c) region of the antibody. Based on this finding, interdomain interactions between Ec DOS and the isolated heme-bound PAS domain were investigated on the solid surface. Ec DOS immobilized via anti-(His) 6 -Tag mAb maintained interactions with the PAS fragment, in contrast to directly immobilized Ec DOS in the absence of anti-(His) 6 -Tag mAb. Heme-redox-sensitive interactions between Ec DOS and the PAS fragment were additionally detected using anti-(His) 6 -Tag mAb as a mediator. Our results collectively suggest that the immobilization method using anti-Tag antibody is suitable for maintaining native protein characteristics to facilitate elucidation of their structures and functions on solid surfaces.

Protein microarray technology is a powerful, popular tool for the high-throughput analysis of protein interactions. One important use for protein microarray technology is protein quantification by immunoassay, which was originally based on enzyme linked immunosorbent assay (ELISA) methods. Recently, new research and diagnostic applications have created a need for a rapid and easily applied high-throughput protein quantification method. Here, we introduce several novel techniques that address these needs. Our improved protein microarray-based sandwich immunoassay techniques allow researchers to: (1) control the size and shape of protein spots on the microarray using a perforated seal; (2) analyze two proteins within a single spot, thus increasing the number of tests run on a single microarray without increasing the number of protein spots; (3) improve the efficiency and speed of the Ag-Ab interaction through vibratory reagent convection, which increased the signal intensity by more than two-fold and decreased the reaction time from 30 to 10 min. These new techniques will facilitate rapid immunoassays for diagnostic purposes and other research areas utilizing protein microarray analysis, such as investigations of ligand-receptor or protein-small molecule interactions.

The Ah receptor (AhR) and HLF are transcription factors involved in xenobiotic metabolism and hypoxic response, respectively. AhR and HLF heterodimerize with Arnt as the common partner, and bind to asymmetric E-boxes termed XRE and HRE, respectively. In order to investigate nucleotide preference of the heterodimers, reporter plasmids with oligonucleotides for XREs or HREs with systematic mutations were constructed and their activity was determined. Comparison of the activity revealed that DNA length and nucleotide preference recognized by Arnt subunit in the two heterodimers were largely different between XRE and HRE. We expressed AhR±Arnt and HLF±Arnt in Escherichia coli and used them for DNA binding. The dissociation constant of HLF±Arnt±HRE was 10.4 T 1.6 nM. Competition activity of mutated XREs or HREs with wild type was consistent with their transcription activity. Bending of XRE and HRE induced by binding of the relevant heterodimers was observed with stronger bending of XRE than of HRE. By deletional and mutational analyses, an alanine and three arginine (Ala 8, Arg 9, Arg 11 and Arg 12) residues in the basic sequence of HLF were found to be indispensable for the transcriptional activity.

The emergence of antimicrobial-resistant (AMR) bacteria poses an imminent threat to our society and requires urgent actions. We here propose that a bacteriophage-derived protein called endolysin can be exploited as a new antimicrobial agent to tackle this global public heath issue. Endolysins are hydrolytic enzymes that cleave bacterial cell-walls (peptidoglycan layers), allowing for rapid cell lysis in a matter of minutes. Not only this swift response makes a stark contrast with conventional antibiotics, but the protein nature of endolysins makes them amenable to modifications. For example, modular domains of endolysin can be recombined to create new artificial endolysins. Using in-house high-throughput screening system, we show that we can quickly discover multiple endolysins against methicillin-resistant Staphylococcus aureus (MRSA) with improved activity compared to previously reported ones. We also discuss the prospects for the development of antimicrobial endolysins against any potential pathogens using our proprietary microbial single-cell genome sequencing platform, bit-MAP (R) .

A series of trans-heteroleptic paddlewheel-type diruthenium(II,II) complexes [Ru 2 (2,6-(CF 3) 2 PhCO 2) 2 (p-RPhCO 2) 2-(THF) 2 ] was synthesized by substituting para-R-substituted benzoate ligands (p-RPhCO 2 ¹ , R = CF 3 , Cl, F, H, Ph, Me, MeO, OH) in [Ru 2 (p-RPhCO 2) 4 (THF) 2 ] with 2,6-bis(trifluoromethyl)benzoate (2,6-(CF 3) 2 PhCO 2 ¹). The redox potential (E 1/2) for the [Ru 2 II,II ]/[Ru 2 II,III ] + couple and the HOMO energy level were linearly dependent on the acidity (pK a) of the substituted benzoic acids (p-RPhCO 2 H) and Hammett constants of p-R substitution groups.