Mechanisms and Structural Transitions of Metal Ion-Induced Silk Fibroin Coacervates = 금속 이온에 의해 유도된 실크 피브로인 코아세르베이트의 형성 기작 및 구조 전이

다국어 초록 (Multilingual Abstract)

Living organisms use liquid–liquid phase separation (LLPS) to organize and transform biological polymers like proteins and polysaccharides into functional materials through controlled self-assembly. Silk fibroin (SF), the core protein constituent of silkworm silks, is known for its strength and biocompatibility. However, efforts to process SF into diverse material forms often rely on chemical modifications or organic solvents that disrupt its natural assembly. In contrast, silkworms utilize aqueous LLPS systems to precisely control the silk spinning process, a mechanism that is still not well understood.
This thesis investigates the LLPS behavior of SF and the morphological transition of SF coacervates. The first part examines how salt ions, particularly calcium ions, promote SF LLPS in macromolecularly crowded environments. Calcium ions were proposed to drive SF LLPS through a combination of two interaction mechanisms: (1) increased hydrophobic interactions via charge screening of the negatively charged domains of SF, and (2) formation of salt bridges between carboxylic residues of SF through electrostatic interactions. The domain-specific interaction of calcium ions was further supported by the disruption of LLPS upon the addition of reducing agents, which detached the L-chain from the H-chain and alter the net charge environment of the C-terminal domain. Furthermore, the morphology of SF coacervates was found to transition into fibrils under acidic conditions and shear stress. This observation enabled a biomimetic dry spinning system for producing silk fibers from an aqueous LLPS solution.
The second part explores how SF coacervates form hollow cavities in response to compositional changes in the LLPS solution. Hollow coacervate formation was found to result from transient, out-of-equilibrium shifts triggered by abrupt dilution. Furthermore, inducing a liquid-to-solid transition in SF using kosmotropic salts led to the conversion of liquid hollow coacervates into solid-like capsule coacervates. These capsule coacervates exhibited molecular weight-dependent permeability and selective partitioning toward enzymes as well as hydrophobic and cationic small molecules. Leveraging these properties, a novel strategy was proposed for developing microcapsules with enhanced structural stability against enzymatic degradation and improved material encapsulation efficiency.
Altogether, this work offers new insights into the physicochemical regulation of SF LLPS and its morphological transitions, drawing inspiration from natural silk spinning processes. The findings contribute to a deeper understanding of protein self-assembly and present a sustainable means of material fabrication.

국문 초록 (Abstract)

자연의 다양한 생명체들은 액체–액체 상분리(LLPS)를 통해 단백질과 다당류와 같은 천연 고분자의 자가 조립을 조절하여 기능성 소재 및 구조체를 형성한다. 누에 실크 섬유의 주요 단백질인 실크 피브로인(SF)은 우수한 강도와 생체적합성으로 잘 알려져 있고, 이를 활용하는 다양한 연구가 진행되고 있다. 그러나 SF를 여러 물질 형태로 가공하는 과정은 많은 경우 화학적 개질이나 유기용매의 사용을 수반하며, 이는 누에가 효율적으로 SF을 섬유화하는 기작과는 다른 접근이다. 누에가 LLPS를 기반으로 SF의 자가조립을 조절하는 기작에 관한 이해는 기존 소재화 공정의 효율성을 높이는 기여를 할 수 있을 것으로 기대되지만, 이에 대한 연구가 부족하다.
본 논문에서는 SF의 LLPS 거동과 코아세르베이트의 형태 전이를 연구하였다. 첫 번째 장에서는 염 이온, 특히 칼슘 이온이 거대분자 밀집 환경에서 SF의 LLPS를 어떻게 유도하는지 살펴보았다. 실험 결과를 토대로, 두 가지 상호작용 기작을 제시하였다: (1) SF의 음전하 작용기의 전하가림 효과 (charge screening)를 통해 소수성 상호작용 증가, (2) SF의 카복실기 사이에서 정전기적 상호작용을 통한 염 다리 (salt bridge) 형성. 또한, 칼슘 이온과 SF이 음전하를 띠는 영역 사이의 특이적 상호작용의 영향은 환원제에 의한 L-chain의 분해가 LLPS 해체로 이어지는 현상을 통해 뒷받침되었다. 더 나아가, SF 코아세르베이트의 형태가 산성화와 전단 응력 조건 하에서 섬유화되는 것을 확인하였고, 이 현상을 토대로 LLPS 수용액에서 생체 모방 건식 방사 시스템 개발이 가능함을 제시하였다.
두 번째 장에서는 LLPS 수용액의 조성 변화가 코아세르베이트의 공동화 현상에 어떻게 기여하는 지 살펴보았다. 중공 코아세르베이트(hollow coacervate)는 급격한 희석 환경에서 일시적인 평형 이탈 현상에 의해 나타나는 구조로 확인되었다. 또한, 코스모트로픽 염(kosmotropic salt)의 첨가로 SF의 액체-고체 전이를 유도함으로써 중공 코아세르베이트를 캡슐 코아세르베이트 (capsule coacervate)로 전환하였다. 캡슐 코아세르베이트는 분자량에 따라 투과도가 달라지고, 효소나 소수성 및 양이온성 분자에 대한 선택적 분배(selective partitioning) 특성을 보였다. 이러한 특성을 활용하여 효소 분해에 대한 높은 구조적 안정성과 효율적으로 물질을 담지할 수 있는 마이크로캡슐 제조 방법을 제시하였다.
본 연구는 실크 방사 과정의 이해를 통해 SF LLPS의 물리화학적 조절과 SF 코아세르베이트의 형태 전이에 대한 새로운 통찰을 제공한다. 또한, 본 연구의 결과는 단백질의 자가조립에 대한 더 깊은 이해에 기여하고, 지속가능한 소재 제작 방법의 개발에 기여할 것으로 기대된다.

목차 (Table of Contents)

• Abstract ⅰ
• Table of Contents ⅲ
• List of Tables ⅷ
• List of Figures ⅸ
• Ⅰ. Introduction 1
• Ⅱ. Literature Survey 3
• 2.1. Liquid–liquid phase separation (LLPS) 3
• 2.1.1. Mechanism 3
• 2.1.2. Properties of coacervates 5
• 2.1.3. Involvement of ionic species in inducing LLPS 5
• 2.1.4. Macromolecular crowding 7
• 2.2. Silk fibers 8
• 2.2.1. Silk fiber and its protein, silk fibroin (SF) 8
• 2.2.2. The silk spinning process of silkworms 9
• 2.2.3. LLPS of SF 11
• 2.3. Microcapsules 13
• 2.3.1. Microcapsule fabrication 13
• 2.3.2. Hollow coacervate transition 14
• Ⅲ. Calcium ion-induced LLPS of SF and the transition of SF coacervates to fibers through acidification and shear stress 16
• 3.1. Introduction 16
• 3.2. Materials and methods 18
• 3.2.1. Preparation of the aqueous silk fibroin solution 18
• 3.2.2. Inducing SF LLPS 18
• 3.2.3. Ion specificity for inducing SF LLPS 19
• 3.2.4. Phase separation diagram 19
• 3.2.5. Fluorescence imaging for LLPS driving force analysis 20
• 3.2.6. Acidification and application of shear force to SF coacervates 21
• 3.2.7. Pultrusion-based dry spinning of SF LLPS solution 21
• 3.2.8. Turbidity measurements and SF concentration calculations 22
• 3.2.9. Separating the coacervates from the dilute phase 23
• 3.2.10. Fourier transform infrared spectroscopy (FTIR) 23
• 3.2.11. Circular dichroism (CD) spectroscopy and thioflavin T (ThT) analyses 25
• 3.2.12. Zeta potential measurements 25
• 3.2.13. Amino acid composition and pI calculation 25
• 3.2.14. Fluorescence recovery after photobleaching (FRAP) analysis 26
• 3.2.15. Scanning electron microscopy (SEM) 28
• 3.2.16. SF molecular weight analysis 28
• 3.2.17. Statistical analysis 29
• 3.3. Results and Discussion 30
• 3.3.1. Calcium ion-induced LLPS of SF 30
• 3.3.2. Phase separation diagram 32
• 3.3.3. Degree of SF condensation within the coacervates 38
• 3.3.4. Secondary structure of SF within the coacervates 41
• 3.3.5. Diffusivity of SF within the coacervates 46
• 3.3.6. Effect of various salt ions in inducing LLPS of SF 49
• 3.3.7. Mechanism of calcium ion-induced LLPS of SF—hydrophobic interaction 55
• 3.3.8. Mechanism of calcium ion-induced LLPS of SF—salt bridge formation 60
• 3.3.9. Involvement of L-chain in calcium-ion induced LLPS of SF 65
• 3.3.10. Effect of acidification and shear force on the morphology of SF coacervates 69
• 3.3.11. Effect of acidification and shear force on the SF within the coacervates 75
• 3.3.12. Pultrusion-based dry spinning of silk fibers from SF LLPS dope solution 78
• 3.4. Summary 84
• Ⅳ. Calcium and copper ion-induced LLPS of SF and the transition of SF coacervates to microcapsules through abrupt composition change 85
• 4.1. Introduction 85
• 4.2. Materials and methods 88
• 4.2.1. Preparation of the aqueous silk fibroin solution 88
• 4.2.2. Inducing SF LLPS 88
• 4.2.3. Phase separation diagram 88
• 4.2.4. Dilution condition that induces hollow coacervate transition 89
• 4.2.5. Preparation of hollow coacervate, capsule coacervate, and microapsule 90
• 4.2.6. Fluorescent labled SF 90
• 4.2.7. Z-stack 94
• 4.2.8. Fluorescence recovery after photobleaching (FRAP) analysis 94
• 4.2.9. Fluorescent labled HRP and GOx 94
• 4.2.10. Permeability and small molecule partitioning 95
• 4.2.11. Mechanism of capsule coacervate transition 96
• 4.2.12. Fourier transform infrared spectroscopy (FTIR) 96
• 4.2.13. Elemental analysis 97
• 4.2.14. Thioflavin T (ThT) assay 97
• 4.2.15. Enzymatic degradation of microcapsules 97
• 4.2.16. Polymer encapsulation within the microcapsules 98
• 4.2.17. Statistical analysis and reproducibility 98
• 4.3. Results and Discussion 99
• 4.3.1. LLPS of SF induced by both calcium and copper ions 99
• 4.3.2. Phase separation diagram 101
• 4.3.3. Hollow coacervate transition via dilution 112
• 4.3.4. Effect of dilution pathway in inducing hollow coacervate transition 112
• 4.3.5. Effect of dilution speed in inducing hollow coacervate transition 117
• 4.3.6. Effect of diluting specific compounds in inducing hollow coacervate transition 123
• 4.3.7. Capsule coacervate transition from hollow coacervates 131
• 4.3.8. Molecular weight-dependent permeability of capsule coacervates 138
• 4.3.9. Small molecule partitioning of capsule coacervates 141
• 4.3.10. Microcapsule fabrication 143
• 4.3.11. Enzymatic degradation of microcapsules 148
• 4.3.12. Polymer encapsulation within the microcapsules 151
• 4.4. Summary 153
• Ⅴ. Conclusion 154
• Ⅵ. References 155
• Ⅶ. Abstract in Korean 169