Section B - Protein Structure

1. Section B - Protein Structure

2. Contents B1 Amino acids Structure, Charged side chains, Polar uncharged side chains, Nonpolar aliphatic side chains, Aromatic side chains B2 Protein structure and function Size and shapes, Primary structure, Secondary structure, Tertiary structure, Quaternary structure, Prosthetic groups, What do proteins do, Domains, motifs, Families and evolution B3 Protein analysis Protein Purification, Protein sequencing, Mass determination, X-ray crystallography and NMR, Functional analysis, Proteomics

3. B1 Amino acids—Structure • a-carbon is chiral(手性；asymmetric) except in glycine (R is H) 2. Amino acids are dipolar ions (zwitterions ,兼性离子) in aqueous solution and are amphoteric. • The side chains(R) differ in size, • shape, charge and chemical reactivity 4. A few proteins contain nonstandard amino acids that are formed by post-translational modification of proline and lysine.

4. B1 Amino acids—Charged side chains • “Acidic” amino acids: containing additional carboxyl groups which are usually ionized Glu Asp • “Basic” amino acids: containing positively charged groups His Arg Lys

5. B1 Amino acids—Polar uncharged side chains • Ser Thr Asp • Gln Cys • Contain groups that form hydrogen bonds with water, hydrophilic.

6. B1 Aminoacids—Nonpolar aliphatic side chains • Gly Ala Val Leu • Ile Met Pro

7. B1 Amino acids— Aromatic side chains • Accounts for most of UV absorbance of proteins at 280 nm hydrophobic （疏水的) Phe Tyr Trp

9. B2 Protein structure and function—Size and shapes • A few thousands Daltons (x 103) to more than 5 million Daltons (x 106) • Some proteins contain bound nonprotein materials (prosthetic groups or other macromolecules), which accounts for the increased sizes and functionalities of the protein complexes.

10. Two broad classes of protein may be distinguished: ① Globular ( most enzymes) ② Fibrous protein ( silk protein, 角蛋白keratin)

11. B2 Protein structure and function—Primarystructure • A peptide bond is a chemical bond formed between two molecules when the carboxyl group of one molecule reacts with the amino group of the other molecule, thereby releasing a molecule of water (H2O).

12. N terminus C terminus Structure of the pentapeptide (胸腺喷丁,促胸腺生成素32-36五肽[免疫调节剂])Ser-Gly-Tyr-Ala-Leu.

13. B2 Protein structure and function—Secondary structure • a-helix: • right-handed 3.6 aa per turn hydrogen bond N-H···O=C

14. b-sheet: • hydrogen bonding of the pepetide bond N-H and C=O groups to the complementary groups of another section of the polypeptide chain

15. B2 Protein structure and function—Tertiary structure • The different sections of a-helix, b-sheet, other minor secondary structure and connecting loops of a polypeptide fold in three dimensions

16. Noncovalent interaction between side chains that hold the tertiary structure together: van der Waals forces, hydrogen bonds, electrostatic salt bridges, hydrophobic interactions Covalent interaction: disulfide bonds Denaturation of protein by disruption of its 2o and 3o structure by heat and extremes of pH will lead to a random coil conformation

17. CAM kinase II B2 Protein structure and function—Quaternary structure • Many proteins are composed of two or more polypeptide chains (subunits). These subunits may be identical or different. The same forces which stabilize tertiary structure hold these subunits together. This level of organization called quaternary structure. Many proteins are composed of two or more polypeptide chains (subunits)

18. （a）Primary stucture

19. Advantages of the quaternary structure: • It allows very large protein molecules to be made, such as tubulin. • It can provide greater functionality to a protein by combining different activities into a single entity. • The interactions between the subunits can often be modified by binding of small molecules and lead to the allosteric effects seen in enzyme regulation.

20. B2 Protein structure and function—Prosthetic groups Coenzymes • vitamins:NAD+ (B3) · NADP+ (B3) · Coenzyme A (B5) · THF / H4F (B9), DHF, MTHF · Ascorbic acid (C) · Menaquinone (K) · Coenzyme F420 • non-vitamins: ATP · CTP · SAM · PAPS · GSH · Coenzyme B · Coenzyme M · Coenzyme Q · Methanofuran · BH4 · H4MPT Organic prosthetic groups • vitamins:TPP / ThDP (B1) · FMN, FAD (B2) · PLP / P5P (B6) · Biotin (B7) · Methylcobalamin, Cobamamide (B12) • non-vitamins: Haem / Heme · Lipoic acid · Molybdopterin · PQQ Metal prosthetic groups • Ca2+ · Cu2+ · Fe2+, Fe3+ · Mg2+ · Mn2+ · Mo · Ni2+ · Se · Zn2+

21. Covalently or noncovalently attached to many conjugated proteins （结合、共轭蛋白）, and give the proteins chemical functionality. Many are co-factors in enzyme reactions. Fig. Since not enough energy is liberated in oxidation of succinate and transfer of e to ubiquinon no free hence no gain in free energy results in no pumping of protons in complex II.

22. B2 Protein structure and function—What do proteins do • Enzymes • Signaling • Transport and storage • Structure and movemrnt • Nutrition • Immunity • Regulation

23. B2 Protein structure and function—Domains, motifs, families and evolution • A protein domain is a part of protein sequence and structure that can evolve, function, and exist independently of the rest of the protein chain.

24. Structural motif, a pattern in a protein structure formed by the spatial arrangement of amino acids • A protein family is a group of evolutionarily related proteins, and is often nearly synonymous with gene family. Pyruvate kinase, a protein from three domains

26. B3 Protein analysis— Protein Purification • Gel filtration chromatography • Ion-exchange chromatography • Isoelectric focusing • Electrophoresis • Hydrophobic interaction chromatography • Affinity chromatography • Overexpression

27. Gel filtration chromatography <size>

28. Ion-exchange chromatography <size and charge>

29. Isoelectric focusing <charge>

30. Electrophoresis <charge>

31. Hydrophobic interaction chromatography <hudrophpbicity>

32. Affinity chromatography < affinity>

33. Example： Escherichia coli T7 promoter lac operator lacI coding sequence His · tag IPTG (a lactose analog) His · tag T7 promoter lac operator Vector Overexpression • In the laboratory, the protein encoded by a gene is sometimes expressed in increased quantity. This can come about by increasing the number of copies of the gene or increasing the binding strength of the promoter region.

34. Peptide sequencing by tandem mass spectrometry - backbone cleavages B3 Protein analysis— Protein sequencing • Protein sequencing : • Determine the primary structure of a protein • The two major direct methods of protein sequencing are mass spectrometry and the Edman degradation reaction.

35. Edman degradation Mechanism • Edaman degradation: • Performed in an automated protein sequencer • Determine thesequence of a polypeptide from N-terminal amino acid one by one. • Expensive and laborious

36. Specific enzyme/ chemical cleavage: • Trypsin cleaves after lysine( K) or arginine ( R) • V8 protease cleaves after glutamic acid (E) • Cyanogen bromide cleaves after methionine (M)

37. Most protein sequences are deduced from the DNA/cDNA sequence Direct sequencing: determine the N-terminal sequences or some limited internal sequence  construction of an oligonulceotide or antibody probe fishing the gene or cDNA

39. B3 Protein analysis— Mass determination • Gel filtration chromatography • Electrophoresis ( SDS-PAGE) • Mass spectrometry

40. Gel filtrationchromatography and SDS-PAGE • Comparing of the unknown protein with a proper standard • Popular SDS-PAGE: cheap and easy with a 5-10% error • SDS: sodium dodecyl sulfate, makes the proteins negatively charged and the overall charge of a protein is dependent on its mass.

41. Mass spectrometry: • Molecules are vaporized and ionized, and the degree of deflection (mass-dependent) of the ions in an electromagnetic field is measured • extremely accurate, but expensive • MALDI can measure the mass of proteins smaller than 100 KDa • Helpful to detect post-translational modification • Protein sequencing: relying on the protein data base

42. B3 Protein analysis— X-ray crystallography and NMR X-ray crystallography • Measuring the pattern of diffraction of a beam of X-rays as it pass through a crystal. The first hand data obtained is electron density map, the crystal structure is then deduced. • A very powerful tool in understanding protein tertiary structure. • Many proteins have been crystallized and analyzed.

43. NMR • Measuring the relaxation of protons after they have been excited by radio frequencies in a strong magnetic field. • Measure protein structure in liquid but not in crystal. • Protein measured can not be larger than 30 KDa.

45. B3 Protein analysis— Functional analysis • Tertiary and quaternary structural determination is still a relatively cost and laborious procedure. • Computational methods will allow the prediction of both structure and possible function from simple amino acid sequence information. • Understanding of the true function of a protein still requires its isolation and biochemical and structure characterization. • Identification of all the other proteins with which a protein interacts in the cell is another important aspect of functional analysis. • If the gene foe the protein can be inactivated by mutagenesis or deleted be recombinant DNA techniques, then the phenotype of the resulting mutant can be studied.

46. B3 Protein analysis— Proteomics • Proteomics is the identification and analysis of the total protein complement expressed by any given cell type under defined conditions. • Two-dimensional Protein Electrophoresis • peptide mass fingerprint

47. Two-dimensional Protein Electrophoresis (2DE)

48. Analysis of peptide mass fingerprint of the tryptic digest of P. falciparum actin I by MALDI mass spectrometry. a. Spectrum (500-3500 Da) with zoom-scan (1850-1990 Da) showing N-terminal fragment at 1872 Da and fragment containing histidine 73 at 1947 Da. b. The table shows selected peptide matches from three experiments. N-terminal fragment and fragment containing histidine 73 are in bold.

50. Multiple choice questions 1. Which of the following is an imino acid? A proline. B hydroxy lysine. C tryptophan. D histidine. 2． Protein family members in different species that carry out the same biochemical role are described as . A paralogs. B structural analogs. C heterologs. D orthologs.