Molecular Cell Biology

1. Molecular Cell Biology Professor Dawei Li daweili@sjtu.edu.cn 3420-4744 Textbook: MOLECULAR CELL BIOLOGY 6th Ed Lodish • Berk • Kaiser • Krieger • Scott • Bretscher •Ploegh • Matsudaira Chapter 3-2 1. Quiz: Select to answer 1 question from p108-109 2. Review Chapter 3 Protein regulation 3. Chapter 3.6-3.7-Protein Techniques and Proteomics 4. Answer Questions: How to Preview and Review

2. 3.4 Regulating Protein Function I:Protein Degradation The Proteasome Is a Complex Molecular Machine Used to Degrade Proteins Figure 3-29 Ubiquitin-and- proteasome-mediated prot- eolysis.

3. 3.5 Regulating Protein Function II:Noncovalent and Covalent Modifications Nonconvalent Binding Permits Allosteric,or Cooperative,Regulation of Proteins Experimental Figure 3-30 Hemoglobin binds oxygen cooperatively.

4. Nonconvalent Binding of Calcium and GTP Are Widely Used As Allosteric Switches to Control Protein Activity Ca2+/Calmodulin-Mediated Switching Figure 3-31 Conformational changes induced by Ca2+ binding to calmodulin.

5. Switching Mediated by Guanine Nucleotide-Binding Proteins Figure 3-32 The GTPase switch.

6. Phosphorylation and Dephosphorylation Covalently Regulate Protein Activity Figure 3-33 Regulation of protein activityby the kinase/ phosphatase switch.

7. Regulation III: Proteolytic Cleavage Activates or Inactivates Proteins Coagulation Factor VIII Activation

8. Protein regulation IV: Sub-cellular location change Protein P retains p38N to the nuclear http://virology.biken.osaka-u.ac.jp/English/BDV.html

9. 3.6 Purifying, detecting, and Characterizing Proteins (p92) Differential Centrifugation Rate-Zonal Centrifugation EXPERIMENTAL FIGURE 3-34 Centrifugation techniques separate paticles that differ in mass or density

10. Electrophoresis SDS-PAGE EXPERIMENTAL FIGURE 3-35 SDS-polyactrylamide gel electrophoresis(SDS-PAGE) separates proteins primarily on the basis of their masses

11. 2-D Gel Electrophoresis EXPERIMENTAL FIGURE 3-36 Two-dimensional gel electrophoresis separates proteins on the basis of charge and mass

12. EXPERIMENTAL FIGURE 3-36 Two-dimensional gel electrophoresis separates proteins on the basis of charge and mass

13. Liquid Chromatography EXPERIMENTAL FIGURE 3-37(a) Three commonly used liquid chromatographic techniques separate proteins on the basis of mass, charge, or affinity for a specific binding partner

14. EXPERIMENTAL FIGURE 3-37 (b) Three commonly used liquid chromatographic techniques separate proteins on the basis of mass, charge, or affinity for a specific binding partner

15. EXPERIMENTAL FIGURE 3-37(c) Three commonly used liquid chromatographic techniques separate proteins on the basis of mass, charge, or affinity for a specific binding partner

16. Western Blotting/Immunoblotting EXPERIMENTAL FIGURE 3-38 Western blotting (immunoblotting) combines several techniques to resolve and detect a specific protein

17. Radio Labeling Radioisotopes are indispensable tools for detecting biological molecules

18. Pulse-Chase labeling Labeling Experiments and Detection of Radiolabeled Molecules EXPERIMENTAL FIGURE 3-39 Pulse-chase experiments can track the pathway of protein modification or movement within cells

19. Mass Spectrometry Mass Spectrometry Can Determine the Mass and Sequence of Proteins EXPERIMENTAL FIGURE 3-40 Molecular mass can be determined by matrix-assisted laser desorption/ionization time-of –flight (MALDI-TOF) mass spectrometry

20. Electrospray Ionization –Ion Trap-Mass EXPERIMENTAL FIGURE 3-41(a) Molecular mass of proteins and peptides can be determinded by electrospray ionization ion-trap mass specrometry

21. EXPERIMENTAL FIGURE 3-41(a) Molecular mass of proteins and peptides can be determinded by electrospray ionization ion-trap mass specrometry

22. LC-MS/MS can determine protein sequence

23. Determine 3-D structure by X-ray Crystallography EXPERIMENTAL FIGURE 3-42(a) X-ray crystallography provides diffraction data from which the three-dimensional structure of a protein can be determined Analysis

24. EXPERIMENTAL FIGURE 3-42(a) X-ray crystallography provides diffraction data from which the three-dimensional structure of a protein can be determined

26. 3.7 Proteomics-Definition and Techniques Advanced Techniques in Mass Spectrometry Are Critical to Proteomic Analysis EXPERIMENTAL FIGURE 3-43 LC-MS/MS is used to identify the proteins in a complex biological sample

27. Identify Proteins: Density Gradient Centrifugation and LC-MS/MS EXPERIMENTAL FIGURE 3-44(a) Density-gradient centrifugation and LC-MS/MS can be used to identify many of the proteins in organilles

28. EXPERIMENTAL FIGURE 3-44(a) Density-gradient centrifugation and LC-MS/MS can be used to identify many of the proteins in organilles

29. KEY CONCEPTS OF SECTION 3.7 Proteomics

30. Key words in chapter 3.6 and 3.7 Isoelectric point(pI) 等电点 Liquid chromatography(LC) 液相色谱 Western blotting Radioisotope 放射性同位素 Pulse-chase 脉冲追踪 Proteomics 蛋白质组学

31. Prepare Student Presentations Select 1 topic The principle and Application 1. Gel Electrophoresis 2. Microarray 3. Immunostaining

32. KEY WORDS Deoxyribonucleic acid(DNA) Ribonucleic acid(RNA) Messenger RNA(mRNA) Transfer RNA(tRNA) Ribosomal RNA(rRNA) Transcription Gene expression Purine Pyrimidine Phosphodiester bond Double helix Base pair complementary 脱氧核糖核酸 核糖核酸 信使ＲＮＡ 转运ＲＮＡ 核糖体ＲＮＡ 转录 翻译 嘌呤 嘧啶 磷酸二酯键 双螺旋 碱基 互补

33. 变性 核酸杂交 小ＲＮＡ ＲＮＡ聚合酶 上游 启动子 转录因子 原始转录的RNA 基因组 操纵子 前体ｍＲＮＡ ＲＮＡ剪接 可变剪接 异构体 反义密码子 外显子 内含子 Denaturation Nucleic acid hybridizaiton Micro RNAs(miRNAs) RNA polymerase Upstream Promoter Transcription factor Primary transcript Genome Operon Precursor mRNAs(pre-mRNAs) RNA splicing Alternative splicing Isoform Anticodon Extron Intron

34. Ribosome Genetic code Reading frame Aminoacyl-tRNA Initiation factors(IFs) Elongation factors(EFs) Release factors(RFs) GTPase superfamily Polyribosome Helicase Leading strand Lagging strand Okazaki fragment Cyclin-dependent kinase Recombination 核糖体 密码子 阅读框 氨酰ｔＲＮＡ 起始因子 延伸因子 释放因子 ＧＴＰ酶超家族 多核糖体 解旋酶 前导链 后随链 冈崎片断 ＣＤＫ 重组

35. Point mutation Meiosis Crossing over Virion Bacteriophage Capsid Nucleocapsid Envelope Plaque assay Lysogeny Retrovirus Reverse transcriptase Provirus Onco-gene 点突变 减数分裂 交换 病毒 噬菌体 衣壳 核衣壳 外壳 噬菌斑试验 溶原现象 反转录病毒 反转录酶 前病毒 致癌基因

36. Discussion: • Answer Questions: • 1. How to Preview and Review • 2. How to Present • Homework: • Review Chapter 4 Concepts p161 (will be tested in Final) • Analyzing the data p161-162 • (These will be tested in Final) • 3. Prepare quiz for Chapter 5 • Next Monday in Class Quiz Chapter 5 questions