Biology subjects

Noji, H.

Publications and source records attributed to Noji, H..

3 recordsLinked to original sources

bioRxiv2020

The six steps of the F1-ATPase rotary catalytic cycle

F1Fo ATP synthase interchanges phosphate transfer energy and proton motive force via a rotary catalysis mechanism. When isolated, its F1-ATPase catalytic core can hydrolyze ATP, rotating its {gamma} rotor subunit. Although previous structural studies have contributed greatly to understanding rotary catalysis in F1, the structure of one major conformational state detected in single-molecule studies, termed the binding dwell state, has not yet been determined. Here, by exploiting a temperature-sensitive F1-ATPase mutant from Bacillus PS3, the structure of this binding dwell state was established together with that of the catalytic dwell state. Each state showed three catalytic {beta} subunits in different conformations, providing the complete set of six {beta} subunit conformational states taken up during catalysis cycle. These structures provide molecular details for the power-stroke conformational change that occurs upon ATP binding and induces a ~80{degrees} {gamma} subunit rotation, as well as a second torque-generating conformational change, triggered by hydrolysis and product release, that produces a ~40{degrees} rotation. This study also identifies a putative phosphate-releasing tunnel that indicates how ADP and phosphate releasing steps are coordinated. Overall these findings provide a structural basis for the entire F1-ATPase rotary catalysis cycle.

biophysics↗

bioRxiv2020

Multi-dimensional digital bioassay platform based on an air-sealed femtoliter reactor array device

Single-molecule experiments have been helping us to get deeper inside biological phenomena by illuminating how individual molecules actually work. Digital bioassay, in which analyte molecules are individually confined in small compartments to be analyzed, is an emerging technology in single-molecule biology and applies to various biological entities (e.g., cells and virus particles). However, digital bioassay is not compatible with multi-conditional or multi-parametric assays, hindering understanding of analytes. This is because current digital bioassay lacks a repeatable solution-exchange system that keeps analytes inside compartments. To address this challenge, we developed a new digital bioassay platform with easy solution exchanges, called multi-dimensional (MD) digital bioassay, and tested its quantitativity and utility. We immobilized single analytes in arrayed femtoliter (10-15 L) reactors and sealed them with airflow. The solution in each reactor was stable and showed no cross-talk via solution leakage for more than 2 h, and over 30 rounds of perfect solution exchanges were successfully performed. To show the utility of our system, we investigated neuraminidase inhibitor (NAI) sensitivity on single influenza A virus (IAV) particles in a multi-conditional assay. We proved that IAV particles show a heterogeneous response to the NAI. Further, to demonstrate multi-parametric assays, we examined the sensitivity of individual IAV particles or model enzyme molecules to two different inhibitors. Our results support that MD digital bioassay is a versatile platform to unveil heterogeneities of biological entities in unprecedented resolution.

biophysics↗

bioRxiv2020

Elucidation and control of low and high active populations of alkaline phosphatase molecules for quantitative digital bioassay

Alkaline phosphatase (ALP), a homo-dimeric enzyme has been widely used in various bioassays as disease markers and enzyme probes. Recent advancements of digital bioassay revolutionized ALP-based diagnostic assays as seen in rapid growth of digital ELISA and the emerging multiplex profiling of single-molecule ALP isomers. However, the intrinsic heterogeneity found among ALP molecules hampers the ALP-based quantitative digital bioassays. This study aims quantitative analysis of single-molecule activities of ALP from Escherichia coli and reveals the static heterogeneity in catalytic activity of ALP with two distinct populations: half-active and fully active portions. Digital assays with serial buffer exchange uncovered single-molecule Michaelis-Menten kinetics of ALP; half-active molecules have halved values of the catalytic turnover rate, kcat, and the rate constant of productive binding, kon, of the fully active molecules. These findings suggest that half-active ALP molecules are heterogenic dimers composed of inactive and active monomer units, while fully active ALP molecules comprise two active units. Static heterogeneity was also observed for ALP with other origins: calf intestine or shrimp, showing how the findings can be generalized across species. Cell-free expression of ALP with disulfide bond enhancer and spiked zinc ion resulted in homogenous population of ALP of full activity, revealing that inactive monomer units of ALP are deficient in disulfide bond formation and zinc ion coordination, and also offering the way to prepare homogenous and active populations of ALP for quantitative digital bioassays of ALP.

biophysics↗