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정형섭,우재성,권희석,류범한 한국구조생물학회 2019 Biodesign Vol.7 No.2
The Falcon 2 camera (Thermo Fisher Scientific Inc., USA) mounted in High Resolution Bio-TEM at Korea Basic Science Institute (KBSI) was upgraded to Falcon 3EC in August 2017 (Jeong et al., 2017). Compared with Falcon 2, Falcon 3EC has better sensitivity and higher frame rate and can be operated in electron counting mode. These changes significantly increase the data quality, thereby increasing the resolution of the 3D structure. In the benchmarking test of this machine, the structure of inhibitor-free beta-galactosidase has been solved at 2.16 Å. Here we introduce main features of the new detector, and discuss about major considerations required for its operation and data collection strategies to achieve the near atomic resolution.
김슬기,김한이,우재성,조현수,정연진,이승택,하남출,Kim, Seul-Ki,Kim, Han-Ie,Woo, Jae-Sung,Cho, Hyun-Soo,Jung, Yun-Jin,Lee, Seung-Taek,Ha, Nam-Chul Korean Society of Life Science 2007 생명과학회지 Vol.17 No.2
단백질 티로신 kinase인 PTK6는 대부분의 유방암에서 과발현되며, 암세포의 증식만을 촉진하는데 역할을 한다. 이 연구에서 PTK6의 활성도메인을 초파리의 peptidoglycan recognition protein (PGRP) -LB 단백질을 퓨전파트너로 사용하여 바큘로바이러스 시스템을에서 과발현하는데 성공하였다. 우리는 PGRP-LB가 바큘로바이러스 시스템에서 잠재적으로 퓨전 단백질로 사용될 수 있는 가능성을 처음으로 발견하였다. 정제된 PTK6단백질은 기존의 박테리아에서 발현된 단백질보다 1.5배 높은 활성을 지녔다. 이 단백질은 PTK6의 분자기전 및 그것의 저해제 개발에 필수적인 결정 구조를 규명하는데 사용될 것이다. PTK6, an intracellular protein tyrosine kinase, is significantly overexpressed in a majority of breast cancers and has a role in promoting the proliferation of the cancer cells, but not of normal cells. Here, we report high-level production of the catalytic unit of PTK6 fused with Drosophila peptidoglycan recognition protein (PGRT)-LB, in the baculovirus system. We first found that the PGRP-LB was potentially useful as a fusion partner to increase the yield of heterologous protein in the baculovirus system. The purified recombinant protein exhibited a 1.5-fold activity with much higher yield than the bacterially-expressed protein. The protein expressed in the baculovirus system will be useful for the crystallization to determine its crystal structure helping understand the molecular mechanism of PTK6 and design its inhibitors.
Rescue of Deleterious Mutations by the Compensatory Y30F Mutation in Ketosteroid Isomerase
차형진,최관용,장도수,김연길,홍비학,우재성,김경태 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.1
Proteins have evolved to compensate for detrimental mu-tations. However, compensatory mechanisms for protein defects are not well understood. Using ketosteroid isome-rase (KSI), we investigated how second-site mutations could recover defective mutant function and stability. Pre-vious results revealed that the Y30F mutation rescued the Y14F, Y55F and Y14F/Y55F mutants by increasing the catalytic activity by 23-, 3- and 1.3-fold, respectively, and the Y55F mutant by increasing the stability by 3.3 kcal/mol. To better understand these observations, we systematically investigated detailed structural and thermodynamic effects of the Y30F mutation on these mutants. Crystal structures of the Y14F/Y30F and Y14F/Y55F mutants were solved at 2.0 and 1.8 Å resolution, respectively, and compared with previoulsy solved structures of wild-type and other mutant KSIs. Structural analyses revealed that the Y30F mutation partially restored the active-site cleft of these mutant KSIs. The Y30F mutation also increased Y14F and Y14F/Y55F mutant stability by 3.2 and 4.3 kcal/mol, respectively, and the melting temperatures of the Y14F, Y55F and Y14F/Y55F mutants by 6.4°C, 5.1°C and 10.0°C, respectively. Compensatory effects of the Y30F mutation on stability might be due to improved hydrophobic interactions because removal of a hydroxyl group from Tyr30 induced local compaction by neighboring residue movement and enhanced interactions with surrounding hydrophobic residues in the active site. Taken together, our results suggest that perturbed active-site geometry recovery and favorable hydrophobic interactions mediate the role of Y30F as a second-site suppressor.