Thermal Stability Improvement with Domain-wise Dissection Strategies of Multidomain Proteins
다중 도메인 단백질의 도메인별 분석 전략을 통한 열 안정성 향상
- 주제(키워드) 도움말 Protein thermal stability , Protein engineering , Glutamine-binding protein , Melting temperature
- 발행기관 강릉원주대학교 일반대학원
- 지도교수 도움말 연영주
- 발행년도 2024
- 학위수여년월 2024. 2
- 학위명 석사
- 학과 및 전공 도움말 일반대학원 KIST강릉분원학.연협동과정
- 세부분야 해당없음
- 실제URI http://www.dcollection.net/handler/kangnung/000000011589
- UCI I804:42001-000000011589
- 본문언어 영어
초록/요약 도움말
Proteins, fundamental to all biological processes, function as catalysts, inhibitors, and sensors, and are pivotal in numerous industrial applications, particularly in the biotechnology and medical sectors. Their roles range from facilitating metabolic reactions to acting as molecular switches in cellular pathways. The complexity and diversity of protein functions are significantly enhanced in multidomain proteins, which exhibit structural and functional plasticity, crucial for the intricate biological processes. Multi-domain proteins, characterized by the presence of two or more distinct functional regions or domains, are central to the adaptive mechanisms of organisms. These proteins have evolved through the combination of different domains, each contributing unique functional attributes. This evolutionary strategy has resulted in proteins that are not only versatile in function but also robust in structure. The inter-domain interactions within these proteins are critical for their stability and function, often dictating the overall protein conformation and activity. A key challenge in the application of proteins in industrial settings, particularly those involving high temperatures or harsh conditions, is their thermostability. Enhancing the thermostability of proteins, especially multidomain ones, is crucial for their effective use in various applications. The complexity of multidomain proteins, where the interplay of domains can complicate stability optimization, poses a significant challenge in this regard. This study addresses the challenge of enhancing protein thermostability with a focus on glutamine-binding proteins (QBP) and other multidomain proteins. Recognizing the intricate nature of these proteins, we employed a novel approach to design domain variants with enhanced thermostability. Our strategy involved targeted modifications at the domain level, hypothesizing that improving the stability of a single domain could significantly enhance the overall stability of the entire multidomain protein. Our findings reveal that this domain-based modular engineering strategy not only simplifies the sequence space for both experimental and computational methods but also provides a potent alternative to traditional techniques for protein thermostability enhancement. By focusing on individual domains, we were able to achieve a more precise and effective enhancement of protein stability, overcoming the limitations posed by the complex inter-domain interactions in multidomain proteins. This approach suggests that targeted modifications at the domain level can lead to more stable and functionally efficient protein variants. Such advancements have broad implications for their practical application in various industries, including but not limited to biotechnology, pharmaceuticals, and environmental engineering.
more목차 도움말
1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Domain detection using motion analysis
2.3. Design for stabilization of proteins
2.4. Gene cloning
2.5. Protein purification
2.6. Measurement of melting temperature with CD spectroscopy
2.7. Structure determination and refinement
2.8. Structure prediction by AlphaFold and structural alignment
3. Results
3.1. Domain-wise analysis of the thermal stability of a glutamine binding protein
3.2. Stabilization of QBP by domain-wise modular engineering
3.3. Structural assessment of the stabilized QBPs
3.4. Domain-based engineering of other proteins
4. Discussion and Conclusion
5. Appendix
6. Reference
