한빛사 인터뷰
- 등록2023-04-21
- 조회1,099
1. 논문관련 분야의 소개, 동향, 전망을 설명, 연구과정에서 생긴 에피소드
I was interested in studying the native structure and function of important drug targets in Mycobacterium tuberculosis, the world’s most lethal bacterium which still takes more than 1 million lives each year. I was drawn to studying native protein structure and function for the following reasons: 1) I have often observed that natively-folded proteins behaved much better than recombinantly-produced proteins, and 2) By studying native structures, you can learn a lot about their native functions! I used chromosomally GFP-tagged polyketide synthase 13 (Pks13) strain of Mycobacterium smegmatis to pulldown and purify the native Pks13 complex for structure determination using cryo-EM. Because Pks13 is a stably-dimerized complex of 400kDa, it was an excellent sample for cryo-EM investigation, and our team resolved dynamic structures up to 1.8 A. From our structures, we showed for the first time, how the acyl-carrier protein (ACP) swings back and forth to translocate substrate molecule from one site of the multi-enzyme complex to another, and elucidated at atomic resolutions the native substrates bound at two active sites. By combining the strengths of native protein purification approach and cryo-EM, we were able to identify native substrates and visualize them inside active sites and also elucidate dynamic structures the multi-enzyme complex undergoes while carrying out its reaction mechanism.
2. 연구를 진행했던 소속기관 또는 연구소에 대해 소개 부탁드립니다.
UCSF Department of Biochemistry and Biophysics was a very ideal place for me to carry out my research described in this paper. Because it is a university hospital, we had a tuberculosis expert (M.D., Ph.D.) who served as my co-mentor for this project. And we had a state-of-the art electron microscopy facility where we collected excellent quality data used in this study.
3. 연구 활동 하시면서 평소 느끼신 점 또는 자부심, 보람
Chromosomally tagging and purifying native complexes from mycobacteria for structural studies was very challenging. At each step, there were many hurdles. One major hurdle was obtaining enough native protein material for structural studies. It led me to solubilize the mycobacterial membrane in detergents and running a gel on detergent-solubilized fractions to identify abundantly-expressed native proteins using mass spectrometry. I worked on several targets simultaneously with very high failure rate, and many of my lab mates were concerned that all of my efforts would not bear fruit and suggested I pursue projects using recombinantly-expressed proteins. But because I had seen the method of native protein pulldown for structural studies work in other studies which shed great functional insight, I was determined to keep pursuing, and in the end, the Pks13 target worked beautifully.
4. 이 분야로 진학하려는 후배들 또는 유학준비생들에게 도움이 되는 말씀을 해 주신다면?
I would say, have a solid foundation in protein biochemistry. For any structural studies, first and foremost obtaining highly-pure and active protein sample is key. Knowing how to keep challenging proteins happy is a very valuable skill to have as this paves the way for structural and functional studies. Without a good sample, you cannot collect good data. Having a solid foundation in protein purification and biochemistry methods is key for success in any structural work.
5. 연구 활동과 관련된 앞으로의 계획이 있으시다면?
I recently accepted an offer for a Research Scientist position at Lawrence Livermore National Laboratory. There, I will continue my interest in infectious diseases research by employing my protein biochemistry and structural biology skillsets.
#Polyketide synthase 13
# tuberculosis
# structural biology
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