Applied Biological Chemistry

The Korean Society for Applied Biological Chemistry (한국응용생명화학회)
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Spectroscopic Analysis of Partially Folded State of Ubiquitin

유비퀴틴 단백질의 부분적으로 폴딩된 구조에 대한 분광학적 분석

  • Park, Soon-Ho (Department of Dentistry, College of Dentistry, Kangnung National University)
  • 박순호 (강릉대학교 치과대학 치의학과)
  • Published : 2003.11.30
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Abstract

Hydrophobic core variant of ubiquitin appeared to have partially folded structure at pH around 2. The intrinsic tryptophan fluorescence emission maximum of this ubiquitin variant at pH 2 showed slight blue shift compare to that of unfolded state, suggesting that some residual tertiary structures remain in this solvent condition. At the same solvent condition, this ubiquitin variant binds with hydrophobic dye, 8-anilinonaphthalene-1-sulfonic acid(AMS), which is known to bind to exposed hydrophobic surface. Furthermore, far-UV circular dichroic spectrum of this ubiquitin variant in the diminished pH was remarkably different from the far-UV CD spectrum of the native state or unfolded state. Based on the molar ellipticity at 220 nm, this ubiquitin variant at pH 2 appeared to have significant amount of secondary structures. All these observations suggest that this ubiquitin variant in the diminished solvent pH has loosely folded hydrophobic core with some secondary structures, which are key features of molten globule conformation. Since molten globule has long been considered as a protein folding intermediate, it is considered that this hydrophobic core variant ubiquitin will serve as a valuable model to study protein folding process.

Hydrophobic core가 변이된 유비퀴틴 단백질이 pH 2 용액에서 보이는 구조적인 특성을 여러 분광학적 방법으로 측정하였다. 낮은 pH값을 갖는 용액에서 이 변이 유비퀴틴의 intrinsic tryptophan fluorescence emission spectrum은 unfolded 상태보다 약간 blue shift되어 있고 또한 그 intensity도 상당히 낮게 나타났다. 이는 이 용액 조건에서 이 변이 유비퀴틴의 삼차구조가 약간 남아 있는 것을 의미한다. 같은 용액에서 이 변이 유비쥐틴의 far-UV circular dichroic spectrum은 native 상태나 unfolded 상태의 spectrum과 현저히 달랐으며 220 nm 에서의 molar ellipticity 값을 통하여 볼 때 pH 2인 용액에서 상당량의 이차구조를 지니고 있었다. 또한 같은 용액에서 이 변이 유비퀴틴은 hydrophobic dye인 8-anilinonaphthalene-1-sulfonic acid(ANS)외 fluorescence emission intensity를 증가시키고 fluorescence emission maximum이 짧은 파장에서 나타나게 하였다(blue shift). 이러한 현상은 pH 2 용액에서 이 변이 유비퀴틴의 hydrophobic core가 느슨하여져서 hydrophobic dye인 ANS가 결합할 수 있는 구조를 띠고 있음을 나타낸다. 이러한 분광학적인 관찰은 이 변이 유비귀틴이 pH 2인 용액에서 상당량의 이차구조를 지니고 있지만 hydrophobic core는 느슨하게 형성된 molten globule과 같은 형태를 지니고 있음을 나타낸다. 이 변이 유비퀴틴의 molten 히obule 형태는 단백질 폴딩 반응의 경로를 연구할 수 있는 좋은 모델이 될 수 있을 것으로 생각된다.

Keywords

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