Exploring the structure and mechanism of formation of an artificial protein capsid, toward the development of a novel redox-responsive nano-carrier system.

2013 Fiscal Year Research-status Report

• Project/Area Number: 25560231
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: MALAY Ali 独立行政法人理化学研究所, Heddle国際主幹研究ユニット, 研究員 (40467006)
• Project Period (FY): 2013-04-01 – 2015-03-31
• Keywords: gold catalysis / gold cluster / cryo EM / capsid / drug delivery / protein structure / bionanotechnology / protein shell

Research Abstract

The goal for the first year was to characterize the mechanism of formation of the TRAP artificial capsid (CLS) and probe the structure.
We have made progress in elucidating the mechanism, in particular showing intermediates in the reaction. In addition we have uncovered a possible mechanism of how Au55 gold cluster can catalyze thiol oxidation reactions in general.
In terms of structural characterization, crystallization trials are still on-going. We also performed cryo-EM single-particle analysis, which yielded 11 angstrom resolution map. The map shows clearly how the individual TRAP rings are arranged to form the artificial capsid. Significantly, the symmetry is apparently different from previously reported natural or artificial protein capsids.

Current Status of Research Progress

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

Reason

Although some difficulties were met with obtaining diffraction-quality crystals, results from other studies I carried out have significantly expanded our level of understanding namely of the mechanism by which Au55-type gold clusters might catalyze disulfide bond formation. Also, from cryo-EM studies we have made considerable progress in elucidating the overall structure of TRAP-CLS.

Strategy for Future Research Activity

Crystallization trials will be pursued in order to obtain high quality diffracting crystals of TRAP-CLS; subsequently diffraction data will be collected and the crystal structure will be solved.
I will also attempt loading the CLS with a variety of cargo molecules (small drug molecules, DNA etc). This may require further engineering of the protein, for example to incorporate a binding site in the inside of the CLS. Once loaded we will test the performance of the CLS in unloading cargo at different conditions, including different redox environments.

Expenditure Plans for the Next FY Research Funding

There are several reasons. First is we decided not to employ a research technician. Second is that we decided for a more equal distribution of funds between the 2 years. For the second year we will purchase more materials than previously expected.
For the second year I will focus on completing the studies started in the first year. The main experimental focus will be obtaining the X-ray crystal structure of the CLS capsid. I plan to publish 3-4 papers based on the results of these investigations, and plan to attend 2-3 research conferences (domestic and international).