Section B 蛋白质 结构 Protein Structure

1. Section B 蛋白质 结构 Protein Structure

2. Central dogma中心法则: DNA > RNA > Protein Nucleic acids:(DNA, RNA): polymer of nucleotides (4 for each) Protein:Polymers of amino acids (20 aa)

3. B1 Amino acids:structure, side chains (charged, polar uncharged, nonpolar aliphatic, aromatic) B2 Protein structure and function: Structure: size and shapes, primarysecondary  tertiary  quaternary, prosthetic groups Domains, motif, and family Function B3 Protein analysis Purification Determine sequence, mass, and structure (X-ray crystallography and NMR)

4. B1 氨基酸 basic structure COO- Common structure of 19 AAs Proline 脯氨酸 Ca H H3N R 亚氨基 • a-carbon is chiral (不对称性) except glycine (R is H) • Both D- and L- stereoisomers(异构体), but only L-isomers are found in proteins • Amino acids are dipolar ions (zwitterions [两性离子]) in aqueous solution and are amphoteric (酸碱两性的) • The side chains (R) differ in size, shape, charge and chemical reactivity • Nonstandard amino acids (> proline脯氨酸and lysine赖氨酸)

5. B1 Amino acids- charged (5) Can form salt bridges, are hydrophilic (亲水) 1. “Acidic” amino acids (2): containing additional carboxyl羧基groups which are usually ionized aspartic acid(Asp, D，天冬氨酸) glutamic acid (Glu, E，谷氨酸)

6. 2. “Basic” amino acids (3): containing positively charged groups e Lysine (Lys, K, 赖氨酸) Arginine(arg, R,精氨酸) d a guanidino group (胍基） Histidine(His, H，组氨酸) The imidazole group (咪唑基) has a pKa near neutrality. This group can be reversibly protonated under physiological conditions, which contribute to the catalytic mechanism of many enzymes.

7. B1 Amino acids- polar uncharged (5) Contain groups that form hydrogen bonds with water, hydrophilic亲水的 Serine (Ser, S，丝氨酸) Contain hydroxyl groups. Threonine (Thr, T，苏氨酸) Asparagine (Asn, N，天冬酰氨) Glutamine (Gln, Q，谷氨酰氨)

8. B1 Amino acids- polar uncharged (5) Cysteine (Cys, C，半胱氨酸) has a thiol (巯醇) group， which is often oxidizes to cystine胱氨酸 x-S-S-x

9. B1 Amino acids- nonpolar aliphatic脂肪族的(7) (hydrophobic疏水) Glycine (Gly, G，甘氨酸) Proline (Pro, P，脯氨酸): imino acid (亚氨基酸) Methionine (Met, M，甲硫氨酸): contains a sulfur atom

10. Alkyl (烷基) side chains Alanine (Ala, A，丙氨酸) Leucine (Leu, L，亮氨酸) Isoleucine (Ile, I，异亮氨酸) Valine (Val, V，缬氨酸)

11. B1 Amino acids- aromatic芳香族(3) Accounts for most of UV absorbance of proteins at 280 nm hydrophobic （疏水的) Phenylalanine (Phe, F，苯丙氨酸) Tyrosine (Tyr, Y，酪氨酸) Tryptophan (Trp, W，色氨酸)

12. Non-standard amino acids(稀有氨基酸): e.g. 4-hydroxyproline(4-羟基脯氨酸), 5-hydroxylysine(5-羟基赖氨酸) in collagen(胶原质) - not encoded, formed by post-translational modification(翻译后修饰)

13. B2 蛋白质的结构与功能 Structure: size and shapes, primarysecondary  tertiary  quaternary, prosthetic groups (辅基，nonprotein molecules of conjugated proteins (共轭蛋白) Domains结构域, motif基序, and family家族 Protein function

14. B2 Protein structure －Sizes • A few thousands Daltons (x 103) to more than 5 million Daltons (x 106) • Some proteins contain bound nonprotein materials (prosthetic groups辅基 or other macromolecules), which accounts for the increased sizes and functionalities of the protein complexs.

15. B2 Protein structure －Shapes Globular proteins: enzymes chymotrypsin (糜蛋白酶) Complementary fit of a substrate molecule to the catalytic site (groove-like) on an enzyme molecule.

16. Fibrous proteins: important structural proteins (silk fibroin, keratin in hair and wools ) Keratin(角蛋白) Protofibril(初原纤维) microfibril(微管) keratin in hair

17. 蛋白质的功能 • 1 催化功能----enzyme 酶 • 2 信号传递---- cell membrane protein • 3 转运与贮存---- hemoglobin transports oxygen • 4 结构与运动---- collagen, keratin, tubulinin cytoskeleton, actin and myosin for muscle contraction • 5 营养----casein (酪蛋白) and ovalbumin(卵清蛋白) • 6 免疫---- antibodies • 7 调节---- transcription factors • 8 抗癌药物----毒蛋白 • 9 支持与保护作用-----毛发的角蛋白

18. B2 Protein structure －Primary Polypeptides多肽contain N- and C- termini and are directional, usually ranging from 100-1500 aa Formation of a peptide bond (shaded in gray) in a dipeptide.

19. N terminus C terminus Structure of the pentapeptide Ser-Gly-Tyr-Ala-Leu.

20. B2 Protein structure －Secondary • a-helix • right-handed • 3.6 aa per turn • hydrogen bond • N-H···O=C A stereo, space-filling representation Collagen胶原质triple helix: three polypeptide intertwined

21. b-sheet: hydrogen bonding of the peptide bond N-H and C=O groups to the complementary groups of another section of the polypeptide chain x Parallel b sheet: sections run in the same direction Antiparallel b sheet: sections run in the opposite direction A stereo, space-filling representation of the six-stranded antiparallel b sheet. fibroin蚕丝蛋白

22. B2 Protein structure － Domains (结构域):, motifs （基序）and families（家族） Domains(结构域): structurally independent units of many proteins, connected by sections with limited higher order structure within the same polypeptide. (Figure) They can also have specific function such as substrate binding

23. Structural motifs （基序）: • Groupings of secondary structural elements that frequently occur in globular proteins • Often have functional significance and represent the essential parts of binding or catalytic sites conserved among a protein family bab motif

24. Protein families （家族）:structurally and functionally related proteins from different sources Motif The primary structures of c-type cytochromes from different organisms 趋异进化----直系同源/共生同源。 趋同进化----无关基因进化至产生具有相似结构和催化 活性 蛋白质。如细菌蛋白水解酶与人的糜蛋白酶。

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

26. B2 Protein structure －Quaternary 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. A stereo, space-filling drawing showing the quaternary structure of hemoglobin血色素 a1-yellow; b1-light blue; a2-green; b2-dark blue; heme亚铁血红素-red back

27. B3 Protein analysis 1.Purification: to obtain enough pure sample for study 2. Sequencing: determine the primary structure of a pure protein sample 3. Mass determination: determine the molecular weight (MW) of an interested protein. 4. X-ray crystallography and NMR: determine the tertiary structure of a given sample.

28. The principal properties of proteins used for purification • Size: gel filtration chromatography 2. Charge: ion-exchange chromatography, isoelectric focusing electrophoresis 3. Hydrophobicity: hydrophobic interaction chromatography 4. Affinity: affinity chromatography 5. Recombinant techniques: involving DNA manipulation and making protein purification so easy

29. gel filtration chromatography凝胶过滤色谱(法)

30. 2. Charge: ion-exchange chromatography(离子交换层析), isoelectric focusing(等电聚焦), electrophoresis(电泳) Isoelectric point (pI): the pH at which the net surface charge of a protein is zero - + - + - + - + - + - - + + - + pH=pI pH<pI pH>pI

31. Ion-exchange chromatography Sample mixture + + + Ion displacing Protein binding Column + anions Column + anions Column + proteins Purified protein

32. + Electrophoresis Protein migrate at different position depending on their net charge

33. Isoelectric focusing A protein will stop moving at position corresponding to its isoelectric point (pI) in a pH gradient gel.

34. 3. Hydrophobicity(疏水性): hydrophobic interaction chromatography Similar to ion-exchange chromatography except that column material contains aromatic(芳香族的) or aliphatic alkyl(脂肪烷基) groups

35. 4. Affinity chromatography 亲合色谱法 • Enzyme-substrate binding Substrate analogs: competitive inhibitors ding • Receptor-ligandbinding d • Antibody-antigen binding

36. 5. Recombinant techniques: • Clone the protein encodinggene of interest in an expression vector with a purification tag(纯化标签) added at the 5’- or 3’ end of the gene • Protein overexpressionin a cell • Protein purificationwith affinity chromatography.

37. Determine the primary structure of a protein:p • Amino acid composition: • Acid treatment to hydrolyze peptide bonds: 6M HCl, 110°C for 24 hrs. • Chromatographic analysis色谱[层]分析 • However, you cannot get the sequence!

38. Protein sequence analysis (1) Sequence: HLMGSHLVDALELVMGDRGFEYTPKAWLV Trypsin T1 HLMGSHLVDALELVMGDR T2 GFEYTPK T3 AWLV V8 V1 HLMGSHLVDALE V2 LVMGDRGFE V3 YTPKAWLV

39. Mass Determination Gel filtrationchromatography and SDS-PAGE • Comparing of the unknown protein with a properstandard • PopularSDS-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.

40. Mass Determination Mass spectrometry质谱分析: • Molecules are vaporized and ionized (by Xe/Ar beam), and the degree of deflection (mass-dependent) of the ions in an electromagnetic field is measured • Extremely accurate (0.01% error), but expensive • ESI (electrospray ionization) and MALDI (matrix-assisted laser desorption/ionization) can measure the mass of proteins smaller than 100 KDa • Protein sequencing: relying on the protein data base • Helpful to detect post-translational modification

41. X-ray crystallography and NMR Determing the tertiary structure (3-D) of a protein 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

42. 基因组(genome): 指单倍体细胞中包括编码序列和非编码序列在内的全部DNA分子。 转录组(transcriptome)：一个细胞在它的生存期或它生存的任何一个时间内基因组转录的全部mRNA。 蛋白质组(proteome): 一个细胞在它的生存期或它生存的任何一个时间内转录表达的全套蛋白。 蛋白质组学（proteomics): 阐明生物体各种生物基因组在细胞中表达的全部蛋白质的表达模式及功能模式的学科。包括鉴定蛋白质的表达、存在方式(修饰形式)、结构、功能和相互作用等。

43. Section C 核酸的性质 C1 Nucleic AcidStructure C2Chemical and PhysicalProperties of Nucleic Acids C3 Spectroscopic and ThermalProperties of Nucleic Acids C4 DNA Supercoiling

44. C1Nucleic AcidStructure Comparisons of names of bases, nucleosides and nucleotides Purine: A & G; Pyrimidine: C & T/U; (deoxy)-ribose,

45. C1Nucleic AcidStructure Nitrogenous bases 含氮碱基 Bicyclicpurines: Monocyclicpyrimidine: Thymine (T) is 5-methyluracil (U)

46. C1Nucleic AcidStructure Nucleosides In nucleic acids, the bases are covalently attached to the 1’ position of a pentose sugar ring, to form a nucleoside Glycosidic (glycoside, glycosylic) bond (糖苷键) R Ribose or 2’-deoxyribose Adenosine, guanosine, cytidine, thymidine, uridine

47. C1Nucleic AcidStructure Nucleotides A nucleotide is a nucleoside with one or more phosphate groups bound covalently to the 3’-, 5’, or ( in ribonucleotides only) the 2’-position. In the case of 5’-position, up to three phosphates may be attached. Phosphate diester二酯bonds 4 6 7 5 5 1 2 2 9 4 1 Deoxynucleotides (deoxyribose containing) Ribonucleotides (ribose containing)

48. C1Nucleic AcidStructure 5’end:not always has attached phosphate groups DNA/RNA sequence: From 5’ end to 3’ end Example: 5’-UCAGGCUA-3’ = UCAGGCUA Phosphodiester bonds 3’ end:free hydroxyl (-OH) group

49. C1Nucleic AcidStructure DNA double helix • Watson and Crick , 1953. • Two separate strands Antiparellel (5’3’ direction) • Complementary(sequence) • Base pairing: hydrogen bonding that holds two strands together Essential for replicating DNA and transcribing RNA • Sugar-phosphate backbones(negatively charged): outside • Planar bases (stack one above the other): inside back

50. 5 5 4 4 6 6 3 3 1 1 2 7 2 7 6 6 5 8 5 1 8 1 9 4 4 9 2 2 3 3 A:T G:C Base pairing via hydrogen bonds