Study of enzymes inside liquid-liquid phase separated crowded droplets

2022 Fiscal Year Research-status Report

• Project/Area Number: 22K15065
• Research Institution: Okinawa Institute of Science and Technology Graduate University
• Principal Investigator: DINDO Mirco 沖縄科学技術大学院大学, タンパク質工学・進化ユニット, 客員研究員 (10933088)
• Project Period (FY): 2022-04-01 – 2024-03-31
• Keywords: enzymes / phase separation / protocells / cytosol fluidification / protocells migration / chemical gradient

Outline of Annual Research Achievements

We recently discovered for the first time that enzymatically active artificial protocells are able to move in solution following a chemical gradient. The pH change generated by the enzyme within the droplets drives the droplets movement in chemotactic manner. We have investigated on the molecular mechanism in detail by means of biochemical, imaging and fluid mechanism studies. We also demonstrated that these droplets are able to reconstitute a simple and linear protometabolic pathway. We are currently studying the catalytic efficiency of several enzymes inside our liquid-liquid phase separated protocells.

Current Status of Research Progress

1: Research has progressed more than it was originally planned.

Reason

The system (liquid-liquid phase separated droplets or protocells) is very well established and very stable. The partitioning of the enzymes is also very good, and for these reasons we have found no problems during the experimental procedures.

Strategy for Future Research Activity

We are investigating on the increased catalytic activity of two oxidoreductases, L-lacatate dehydrogenase (LDH) and alcohol dehydrogenase (ADH) inside the droplets. Lastly, we are also studying the effect of the high metabolic activity on the physical state of the biological matter (high viscosity environment).We are in the final stage of the project. We are investigating the physical state of the droplets under high metabolic activity (high enzyme concentration). In detail, we are using a highly crowded protein environment inside the droplets and after the activation of the enzyme within the droplets we study the fluidification of the system using FRAP (Fluorescence Recovery After Photobleaching).

Causes of Carryover

We are planning to finish the experiments with the carry-over which will be used to buy the last reagents to finish the experiments, for traveling and to cover the expenses to present the data at the conferences and for the publication fees (one article has been submitted and another in preparation)