https://doi.org/10.1101/2025.03.29.646054
요약
- 양전하 아미노산의 종류와 단백질 순전하(net charge)가 condensate 형성 및 물성을 결정: Arginine과 lysine을 포함한 engineered GFP 변이체를 통해 RNA와의 상분리(condensate formation)에 있어 아미노산 종류와 net charge가 상분리 경계, 소금 저항성(salt resistance), 단백질 이동성에 영향을 준다는 것을 규명하였습니다.
- Arginine이 lysine보다 상분리 촉진 및 salt stability 증가: 동일한 net charge 조건에서도 arginine-tagged 단백질은 lysine-tagged 단백질보다 상분리 경계가 확장되고, NaCl 농도 증가에도 더 높은 안정성을 보였습니다. 이는 arginine의 guanidinium group이 π-π 및 cation-π 상호작용을 가능하게 하여 electrostatic interaction을 강화하기 때문입니다.
- 세포 내 (E. coli) 상분리에서도 arginine 효과 확인: E. coli 내에서 arginine-tagged 단백질은 lysine-tagged 단백질보다 condensate 내 농도 분포(partitioning)가 높고, 형성된 condensate 수와 크기도 컸습니다. 이는 in vitro 결과와 일치하며, arginine이 세포 내 상분리에도 더 유리하게 작용함을 보여줍니다.
- FRAP으로 확인한 protein mobility 차이: Arginine-tagged condensate는 lysine-tagged condensate보다 fluorescence recovery after photobleaching (FRAP)에서 회복 속도가 느리고 이동성이 낮음을 확인했습니다. 예를 들어, GFP(+6)-6Arg는 50% 회복에 59초, GFP(+6)-6Lys는 18초가 걸려 3배 이상의 차이를 보였습니다.
- condensate 내부 RNA 포함과 DNA 배제 확인: mPepper aptamer와 DAPI 염색을 통해 condensate 내부에는 RNA가 포함되고 DNA는 배제되는 것을 시각화했습니다. Cryo-electron tomography (cryo-ET)를 통해 condensate 내부가 amorphous-like density를 가지며, 이는 RNA 및 리보솜 포함 가능성을 시사했습니다.
Abstract
Understanding the formation of biomolecular condensates (BMC) in biological systems has proven to be a paradigm shift in our understanding of the subcellular organization of biomacromolecules. From RNA metabolism, stress response mechanisms, and amyloidogenic pathologies, condensates have been implicated to play a role in a myriad of cellular phenomena. Despite their near ubiquity, we still do no wholly understand how the primary sequence of biomolecules influences their biophysical and rheological properties. Here, we aim to understand the impact of primary cationic amino acid composition on the properties of condensates. Using engineered recombinant proteins, we show that the formation and phase boundaries of coacervates formed between proteins and RNA is dependent on the cationic amino acid identity, as well as the net charge of the protein involved in condensation. Despite the equivalent charge between arginine and lysine at physiological pH, arginine has been shown to promote increased encapsulation efficiency and salt stability, as well as reduced protein mobility within condensates. We show that arginine-tagged globular proteins also have a higher salt resistance in vitro when compared to similar lysine-tagged globular proteins. This translates to a cellular context in which arginine tagged proteins promote increased condensate formation in model E. coli cells. We were also able to observe a reduction in the total fluorescent recovery and protein mobility within arginine-based condensates via FRAP. Together, these results suggest that in addition to electrostatic interactions and disorder as the main driving forces of phase separation in biological contexts, the primary sequence and side chain composition of proteins plays a significant role in dictating dynamics of coacervates.