A High-Throughput Assay Platform for Next-Generation Mechanistic Enzymology and Applications
CELL PRESS. 2020: 535A
View details for Web of Science ID 000513023203420
- A Microfluidics-Based Assay for Mapping Connectivity in Highly Proficient Enzymes Reveals Functional Modularity CELL PRESS. 2019: 66A
- Bringing Enzymology into the Genomic Era: Developing and Deploying New Tools to Quantitatively Map Functional Connections Throughout an Enzyme CELL PRESS. 2019: 23A
Role of Polyubiquitin Chain Flexibility in the Catalytic Mechanism of Cullin-RING Ubiquitin Ligases
JOURNAL OF PHYSICAL CHEMISTRY B
2019; 123 (4): 776?86
Cullin-RING ubiquitin ligases are a diverse family of ubiquitin ligases that catalyze the synthesis of K48-linked polyubiquitin (polyUb) chains on a variety of substrates, ultimately leading to their degradation by the proteasome. The cullin-RING enzyme scaffold processively attaches a Ub molecule to the distal end of a growing chain up to lengths of eight Ub monomers. However, the molecular mechanism governing how chains of increasing size are built using a scaffold of largely fixed dimensions is not clear. We developed coarse-grained molecular dynamics simulations to describe the dependence of kcat for cullin-RING ligases on the length and flexibility of the K48-linked polyUb chain attached to the substrate protein, key factors that determine the rate of subsequent Ub attachment to the chain, and therefore, the ensuing biological outcomes of ubiquitination. The results suggest that a number of regulatory mechanisms may lead to variations in the rate of chain elongation for different cullin-RING ligases. Specifically, modulation of the distance between the target lysine and the phosphodegron sequence of the substrate, the distance between the substrate lysine and the active site cysteine of the Ub conjugation enzyme (E2) bound to the cullin-RING scaffold, and flexibility of the bound E2 can lead to significant differences in the processing of K48-linked chains on substrates, potentially leading to differences in biological outcomes.
View details for DOI 10.1021/acs.jpcb.8b10706
View details for Web of Science ID 000457816300004
View details for PubMedID 30590923
An Open-Source, Programmable Pneumatic Setup for Operation and Automated Control of Single- and Multi-Layer Microfluidic Devices.
2018; 3: 117?34
Microfluidic technologies have been used across diverse disciplines (e.g. high-throughput biological measurement, fluid physics, laboratory fluid manipulation) but widespread adoption has been limited in part due to the lack of openly disseminated resources that enable non-specialist labs to make and operate their own devices. Here, we report the open-source build of a pneumatic setup capable of operating both single and multilayer (Quake-style) microfluidic devices with programmable scripting automation. This setup can operate both simple and complex devices with 48 device valve control inputs and 18 sample inputs, with modular design for easy expansion, at a fraction of the cost of similar commercial solutions. We present a detailed step-by-step guide to building the pneumatic instrumentation, as well as instructions for custom device operation using our software, Geppetto, through an easy-to-use GUI for live on-chip valve actuation and a scripting system for experiment automation. We show robust valve actuation with near real-time software feedback and demonstrate use of the setup for high-throughput biochemical measurements on-chip. This open-source setup will enable specialists and novices alike to run microfluidic devices easily in their own laboratories.
View details for PubMedID 30221210