Protein Engineering 서울대학교 화학생물공학부 백 승 렬

단백질 공학: 단백질의 변형을 통하여 유용성이 뛰어난 신기능성 단백질의 생산 유전공학: 생물공학의 제1의 물결 [다량의 단백질 제공] 단백질공학: 생물공학의 제2의 물결 [산업적 효용가치 증대] 따라서, 생명과학 관련 다양한 분야의 지식과 기술의 접목 필요성

Central Dogma

Cycle of Protein Engineering

돌연변이 유도: [1] rational design: site-directed mutagenesis [2] non-rational (random) design: DNA shuffling or directed evolution Structure-Function Relationship of Protein 단백질의 변형을 통하여 다양한 성질의 변화를 추구함. Examples [1] 산업적 효소: protease, glucose isomerase, calf chymosin, papain, amylase, cellulase 및 xylanase, pectinolytic enzyme, lipase 등 [2] 의료용 단백질: 다양한 생리활성의 펩타이드, peptide 또는 polypeptide vaccine, antibody, 세포내 신호전달 관련 단백질, 유전자 발현 관련 단백질, hormone과 수용체 등 [3] 환경산업관련 효소

Biological Structures = Nanomaterials

Top-down Bottom-up Life Biological Phenomena Molecular Interactions Individual Biomolecules [Proteins/Nucleic acids/CHO/Lipids]

전체 > S 부분 (open system) Complexity Bottom-up approach와 Top-down approach사이의 적절한 조화

Lipids Fatty acids

Central Dogma in Biological System Transcription Translation Posttranslationally modified proteins Protein-ligand interactions DNA RNA Protein Reverse Transcription Information Function Information Function Biological phenomena

Proteomics (St./Fn.) (Structural) Functional Genomics Postgenomic era 단백질의 특성 및 기능 파악 조직별 5,000 - 20,000 추정 DNA 염기서열 약 30억개 (2-3% 유전자) 유전자 및 기능파악 총 4 만 유전자 추정 현재까지 9,000여종 파악

[Amino acids]

[Nonstandard amino acids]

[Peptide]

Peptide의 생리활성 Aspartame: aspartate와 phenylalanine Oxytocin: 자궁수축 Bradykinin: 항염증효과 Enkephalin: 마취성, 통증조절 Insulin, glucagon, somatostatin: Glucose metabolism Tissue specific limited proteolysis of precursor polypeptides (in vivo) Solid phase synthesis (in vitro)

[Protein] Noncovalent interactions Ionic interaction Hydrogen bond Hydrophobic interaction van der Waals interaction Structural flexibility – Functional variety Protein folding : Entropy

[Structural hierarchy of protein]

[Denaturation-Renaturation Experiment by Christian Anfinsen]

Levinthal’s paradox Folding mechanisms Diffusion-collision (microdomain) model Framework model Hydrophobic collapse model Molten globule model Jigsaw puzzle model

[Hydrophobic collapse model]

[Molecular Chaperone] Holdase vs. Foldase

Creutzfeldt-Jakob disease [Spongiform encephalopathies] degradation Misfolded protein Folded state Amino acids refolding Accumulation Amyloid formation Degenerative disorders Creutzfeldt-Jakob disease [Spongiform encephalopathies]

Amyloidogenesis

Neurodegenerative disorders The amyloid formation is the common phenomenon observed in various neurodegenerative disorders, including Parkinson’s disease, Alzheimer’s disease, Huntington’s chorea, Amyotrophic lateral sclerosis, Prion disease, etc. Parkinson’s disease Prion disease [mad cow disease] Alzheimer’s disease

[Enzyme] Substrate specificity (기질특이성) Acceleration of chemical reaction (반응가속화) Mild conditions (온순조건) (37 degree and pH 7.4) Rate Enhancement (반응속도 증가) without affecting Chemical Equilibrium (반응평형) Binding Energy between Enzyme and Substrate

Most biomolecules are quite stable. No reaction w/o enzyme Enzyme: Active site + Other region

Enzyme catalyzed reaction: 반응평형 무관, 반응속도만 증가 increase reaction rate without affecting chemical equilibrium cf) mass action ratio Go’ = - RT ln Keq vs. k = kT/h exp (- G‡/RT)

Protein-based Nanobiotechnology

[Protein-based Suprastructure Formation]

Proteins in Nanodevices 1. Motor proteins 2. Chemical sensing built-in switch 3. Cargo delivery using protein transporters and pores 4. Chemical transformation [enzyme catalysis] 5. ATP as energy source [ATP generation system] 6. Data processing [DNA computing]

Protein Engineering (1) Novel and altered functions (2) Physical properties such as enhanced stability (3) Structure based materials Enzymes for pharmaceutical and biotechnology industries Proteins as materials (1) Nanoscale architecture (2) Rich chemistry (3) Versatile enzymatic activities Advances in protein engineering (1) de novo protein design (2) molecular biology (3) non-natural amino acids and peptide ligation (4) protein assembly

Natural proteins as materials [1] Flagellar motors of bacteria

[2] Linear motors of muscle and cytoskeleton

[3] Bacteriorhodopsin

Bacteriorhodopsin Film Photo-rewritable information storage devices Photochromic ink

Proton Gradient and ATP Synthesis FoF1-ATPase

Bacteriorhodopsin and FoF1-ATPase

[4] Molecular Motor

Proteins as novel materials Self-assembly Recognition specificity Monodispersity Biofabrication Proteins as important design elements in the development of advanced materials Protein stability Fusion materials

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