Investigating Amino acid Enrichments and Patterns in Phase-Separating Proteins: Understanding Biases in Liquid-Liquid Phase Separation

https://doi.org/10.1101/2024.12.19.629394

 

Abstract

Liquid-Liquid Phase Separation (LLPS) forms membraneless organelles, enhancing biochemical processes. The stickers-and-spacers model explains LLPS but is mainly validated in Prion-like RNA Binding Proteins. We explore peptide motifs in LLPS in broader protein contexts. We developed a computational approach for motif discovery, implemented in 178 Phase-Separating Proteins (PhSePs), complemented by the FuzDrop and CIDER servers, which identified droplet-promoting regions (DPRs) and examined disorder-related characteristics. Our database of PhSePs was analyzed against proteins with low propensity for LLPS. This comparative analysis revealed 129 enriched peptide motifs with folds higher than 0.2, consisting of 3 to 6 residues, with tetrapeptides being the most prevalent. Key features of the enriched motifs included Gly-rich sequences punctuated with aromatic, charged, and polar residues, as well as homopeptide repeats (e.g., GGDR, SRGG, YGGG, QQQQ, PPPP). Analysis of motif presence, frequency, and co-occurrence revealed widely distributed motifs across different DPRs, identified motifs with significant repetitive patterns, and highlighted motif trios that are more likely to co-occur within a sequence. By harvesting this analysis, we developed a data-driven approach for minimalistic peptide design with LLPS propensity, further using the CIDER server for peptide characterization and peptide design refinement. We designed 8 peptides with various motif combinations and amino acid distributions, which were experimentally validated to undergo LLPS, exhibiting liquid-like behavior with diverse molecular mobility patterns and droplet dynamics. Our approach bridges a non-biased computational approach with experimental validation, offering insights into sequence determinants of phase separation, with the potential for designing minimalistic synthetic condensates with tailored properties.

 

요약

1. 주제 및 배경: 무막 소기관(membraneless organelles, MLOs)은 액-액 상분리(LLPS, Liquid-Liquid Phase Separation)에 의해 형성됩니다. 본 연구는 상분리 단백질(PhSePs)의 아미노산 조성과 패턴을 분석하여 LLPS를 유도하는 서열 특징을 규명하였습니다.
2. 상분리 단백질(PhSePs) 데이터베이스 구축: 178개의 상분리 단백질을 분석하여 FuzDrop 및 CIDER 서버를 이용해 LLPS를 촉진하는 영역(Droplet Promoting Regions, DPRs)을 확인하였습니다.
3. LLPS와 관련된 아미노산 모티프 식별: LLPS를 촉진하는 129개의 아미노산 모티프(3~6개 잔기로 구성됨)를 발견하였으며, 특히 Gly-풍부 서열과 방향족, 전하, 극성 잔기가 혼합된 패턴이 중요한 역할을 한다는 점을 확인하였습니다.
4. 최소 서열 기반 LLPS 펩타이드 설계: 발견된 모티프를 기반으로 8개의 짧은 합성 펩타이드를 설계 및 실험 검증하여, LLPS를 유도하는 서열적 특징을 규명하였습니다.
5. 연구 의의 및 전망: 본 연구는 LLPS를 유도하는 서열적 규칙을 정량적으로 분석하고, 이를 활용한 최소형 LLPS 펩타이드 설계 방법을 제시하였으며, 향후 합성 생물학 및 신약 개발에 기여할 수 있습니다.