Chapter 3 Amino Acids, Peptides, and Proteins

1. Chapter 3 Amino Acids, Peptides, and Proteins

2. Transcription Translation • Posttranslationally • modified proteins • Protein-ligand • interactions DNA RNA Protein Reverse Transcription Biological phenomena Central Dogma in Biological System Specific covalent sequences of monomers Noncovalent Interactions 1. Hydrogen bonds 2. Ionic Interactions 3. Hydrophobic Interactions 4. van der Waals interactions Correct three-dimensional structures Biological functions

3. Amino Acids

4. 1 2 a Amino Acids • 20 Amino acids • Numbering of carbons • a , b, g… from C bonded to NH3+ and COO- • Chiral molecule (exp. Gly) • a-Carbon is a chiral center • Stereoisomer; enantiomer

5. Classification of Amino Acids UV absorption at 280 nm

6. Absorption of light by molecules

7. Classification of Amino Acids • Nonpolar • Structural role

8. Classification of Amino Acids

9. Uncommon Amino Acids • Cell wall (plant) • Collagen (connective tissue) • Collagen • Myosin • A few proteins • Incorporation during translation • Prothrombin (blood clotting) • Ca2+ binding proteins • Elastin

10. Reversible amino acid modifications

11. Amino Acids as Acids and Bases • Zwitterion • Either acids or bases • Ampholytes (substances with zwitterionic nature)

12. Titration of Amino Acids • Two pKa and two buffering regions • pI (isoelectric point or isoelectric pH) • Characteristic pH with zero net electric charge • Above pI : negative charge • Below pI : positive charge • pI = (pK1 + pK2)/2 = 5.92

13. Effect of Chemical Environment on pKa

14. Amino Acids with Ionizable R Group • pI = (pK1 + pKR)/2 = 3.22 • pI = (pKR+ pK2)/2 = 7.59

15. Peptides and Proteins

16. Peptides and Proteins • Peptide; Chains of amino acids • Dehydration reaction b/t amino acids peptide bond • Polypeptide vs. protein • Polypeptide: Mr<10,000 • Amino-terminal (N-terminal) • Carboxyl-terminal (C-terminal)

17. Ionization of Peptide • Ionization of peptide • One free a-amino group • One free a-carboxyl group • Inonizable R groups • pKa of R groups in peptide • Different from pKa of free amino acid  affected by environmental factors

18. Biologically Active Peptides and Polypeptides • Size • Small peptide • Vertebrate hormones (peptide hormones) • Oxytocin (9), thyrotropin-releasing factor (3), insulin (30 + 21) • Antibiotics, amanitin • Most of the proteins • < 2,000 a.a. (exception, titin) • Oligomeric status • Single polypeptide chain • Multisubunit proteins (non-covalent interaction) • Oligomeric : at least two subunits are identical • Protomers : identical units • Calculation of the number of amino acid residues • Mr / 110 • Average Mr of 20 a.a. : 138 • Average Mr of protein a.a : 128 • Removal of water during peptide bond formation : 128 -18 =110

19. Hemoglobin a2b2

20. Conjugated Proteins [1] Simple Protein amino acids [2] Conjugated Protein amino acids + prosthetic groups

21. Working with Proteins

22. Protein Purification • Cell lysis (optionally differential centrifugation) • Crude extract • Fractionation • Use differences in protein solubility • Depending on pH, temperature, salt concentration etc. • Salting out • Addition of ammonium sulfate ((NH4)2SO4) for differential precipitation of proteins • Dialysis • Exchange of salts and buffer using semipermeable membrane (e.g. removing (NH4)2SO4) • Column chromatography • Separation of proteins based on charge, size, binding affinity etc. • Stationary phase vs. Mobile phase containing proteins

23. Ion-exchange chromatography • Cation-exchange chromatography • Solid phase : negatively charged group • Positive charged proteins migrate slowly • Anion-exchange chromatography • Solid phase : positively charged group • pH & salt concentration  affect protein affinity on solid matrix • Separation by pH or salt gradient • Other cautions • Diffusional spreading  expansion of protein band

24. Size-Exclusion Chromatography • Solid phase : cross-linked polymer beads with engineered pores or cavities of a particular size • Small proteins enter the pores • Slow migration

25. Affinity Chromatography • Beads with covalently attached chemical group • Binding of proteins with affinity for the chemical group

26. Protein Purification • HPLC (high-performance liquid chromatography) • Use high pressure pump that speed the movement of the protein molecules • Limited diffusion  High resolution • Determining the methods for protein purification • Mostly empirical

27. Separation & analysis of Protein by Electrophoresis • Electrophoresis • Separation of charged proteins in an electric field • Electrophoretic mobility of proteins • Depending on size and shape of proteins • Advantages • # of different proteins, purity of protein preparation, determination of pI & mw • SDS-polyacrylamide gel electrophoresis

28. Determining Molecular Weight of a Protein SDS PAGE (polyacrylamide gel electrophoresis)

29. Isoelectric Focusing • Procedure to determine the pI of a protein • Establishment of pH gradient • Gel containing a mixture of low molecular weight organic acids and bases (ampholytes) with different pI value • Application of electric field • Each protein migrates until it reaches the pH corresponding to its pI

30. Two-Dimensional Electrophoresis • 1st : Isoelectric focusing • 2nd : SDS-PAGE

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

32. Proteomics [1] Biological Perturbation [2] High-throughput Screening two-dimensional gel electrophoresis in-gel protein digestion MALDI-TOF mass spectrometry peptide mass mapping [3] Bioinformatics [4] Conventional Protein Chemistry

33. Investigation of proteins with Mass spectrometry • Components of mass spectrometer • Ionizer: converting molecules to gas phase ions • Soft ionizer for large molecules • MALDI (matrix-assisted laser desorption/ionization) MS • ESI (electrospray ionization) MS • Mass analyzer: separate the ions according to the m/z • Time of flight (TOF) • Measuring the time take by ions to travel to the detector • Ion detector • Mass spectrometer for protein analysis • Small amount of protein (extraction from 2D-gel) • Determination of molecular weight • Determination of short polypeptide sequence • Tandem MS or MS/MS

34. MALDI-TOF • Matrix-assisted laser desorption/ionization mass spectrormetry • Protein placed in a light-absorbing matrix • Ionization and desorption of proteins by a short pulse of laser

35. ESI-TOF • Electrospary ionization mass spectrometry • Passing of analyte solution through a charged needle with a high electrical potential • Dispersion of charged microdroplets (fine mist)

36. Tandem MS

37. The Structure of Proteins

38. Determination of Amino Acid Sequence • Determination of amino acid sequence from a protein • Sanger’s method • N-terminal labeling and identification • Using FDNB (1-fluoro-2,4-dinitrobenzene) • Edman Degradation • Sequencing of the entire polypeptide • Sequential labeling and removal of the N-terminal amino acid • Sequenator • Automated sequencing of proteins • Accuracy is depending on the efficiency of the individual chemical step • > 99% in modern sequenator • Translation from DNA sequence • DNA sequence  protein sequence • Protein sequence  cloning of the gene

39. Determination of Amino Acid Sequence Sanger Edman

40. Sequencing Large Proteins • Breaking disulfide bonds • Oxidation by performic acid • Reduction and carboxymethylation • Cleaving the polypeptide chain • Using proteases • Cleavage of peptide bond next to particular amino acid residues • Trypsin: Lys, Arg • Sequencing of peptides • Ordering peptide fragments • Compare sequences generated from different cleavage methods • Locating disulfide bonds • Comparison of cleavage fragment with or without breaking disulfide bonds

41. Breaking Disulfide Bonds

42. Sequencing Large Proteins

43. Chemical Synthesis of Small Peptide • Developed by R. Bruce Merrifield (1962) • Synthesis from C- to N- terminal on an polymer support • Fmoc (9-fluorenylmethoxycarbonyl) • Protection of unwanted reaction

44. Chemical Synthesis of Small Peptide

45. Chemical Synthesis of Small Peptide