111生物化學(xué)理論課件@中科大01緒論2008

1、生物化學(xué)生物化學(xué)Biochemistry周叢照周叢照 李衛(wèi)芳李衛(wèi)芳2008年年9月月1日日緒論緒論大綱：課程主要內(nèi)容及要求大綱：課程主要內(nèi)容及要求引子：生物化學(xué)的歷史和發(fā)展趨勢引子：生物化學(xué)的歷史和發(fā)展趨勢教材：教材：生物化學(xué)生物化學(xué)，第三版，王鏡巖等主編，第三版，王鏡巖等主編， 2002年，高教出版社年，高教出版社參考書：參考書：1. Biochemistry, Jeremy M. Berg John L. Tymoczko Lubert Stryer 5th edition W. H. Freeman and Company2. Biochemistry, Donald Voet, Jud

2、ith G. Voet. 3rd edition, 2003, John Wiley & Sons, Inc. New York. 生物化學(xué)（上）生物化學(xué)（上） 5454學(xué)時學(xué)時第一章第一章 緒論緒論 (2(2學(xué)時學(xué)時) ) （周叢照）（周叢照）第二章第二章 蛋白質(zhì)蛋白質(zhì) (16(16學(xué)時學(xué)時) ) （周叢照）（周叢照） 2.1 202.1 20種氨基酸的結(jié)構(gòu)和性質(zhì)（種氨基酸的結(jié)構(gòu)和性質(zhì)（2 2學(xué)時）學(xué)時） 2.2 2.2 蛋白質(zhì)中的共價結(jié)構(gòu)（蛋白質(zhì)中的共價結(jié)構(gòu)（2 2學(xué)時）學(xué)時） 2.3 2.3 蛋白質(zhì)的高級結(jié)構(gòu)（蛋白質(zhì)的高級結(jié)構(gòu)（2 2學(xué)時）學(xué)時） 2.4 2.4 血紅蛋白的結(jié)構(gòu)與

3、功能（血紅蛋白的結(jié)構(gòu)與功能（2 2學(xué)時）學(xué)時） 2.5 2.5 維持蛋白質(zhì)高級結(jié)構(gòu)的作用力（維持蛋白質(zhì)高級結(jié)構(gòu)的作用力（2 2學(xué)時）學(xué)時） 2.6 2.6 蛋白質(zhì)折疊和結(jié)構(gòu)進化蛋白質(zhì)折疊和結(jié)構(gòu)進化 （2 2學(xué)時）學(xué)時） 2.7 2.7 蛋白質(zhì)分離純化（蛋白質(zhì)分離純化（4 4學(xué)時）學(xué)時） 第三章第三章 核酸核酸 （ 6 6學(xué)時學(xué)時) ) （李衛(wèi)芳（李衛(wèi)芳） 3.1 3.1 核苷酸的結(jié)構(gòu)和性質(zhì)核苷酸的結(jié)構(gòu)和性質(zhì) （2 2學(xué)時）學(xué)時） 3.2 3.2 核酸的雙螺旋結(jié)構(gòu)核酸的雙螺旋結(jié)構(gòu) （1 1學(xué)時）學(xué)時） 3.3 3.3 維持核酸高級結(jié)構(gòu)的作用力（維持核酸高級結(jié)構(gòu)的作用力（2 2學(xué)時）學(xué)時） 3.4

4、 3.4 超螺旋結(jié)構(gòu)（超螺旋結(jié)構(gòu)（1 1學(xué)時）學(xué)時）第四章糖（第四章糖（4 4學(xué)時）學(xué)時） （李衛(wèi)芳）（李衛(wèi)芳）4.14.1糖的生物學(xué)作用（糖的生物學(xué)作用（1 1學(xué)時）學(xué)時）4.24.2單糖和多糖（單糖和多糖（2 2學(xué)時）學(xué)時）4.34.3糖蛋白（糖蛋白（1 1學(xué)時）學(xué)時） 第五章第五章 脂類和生物膜（脂類和生物膜（6 6學(xué)時）學(xué)時） （李衛(wèi)芳）（李衛(wèi)芳）5.15.1脂的分類和性質(zhì)（脂的分類和性質(zhì)（2 2學(xué)時）學(xué)時）5.25.2生物膜（生物膜（2 2學(xué)時）學(xué)時）5.35.3膜蛋白（膜蛋白（2 2學(xué)時）學(xué)時）第六章第六章 酶（酶（1414學(xué)時）學(xué)時） （李衛(wèi)芳）（李衛(wèi)芳） 6.16.1酶的作用特

5、征（酶的作用特征（2 2學(xué)時）學(xué)時） 6.26.2酶的分類（酶的分類（1 1學(xué)時）學(xué)時） 6.36.3酶活力測定（酶活力測定（2 2學(xué)時）學(xué)時） 6.46.4酶促反應(yīng)動力學(xué)（酶促反應(yīng)動力學(xué)（3 3學(xué)時）學(xué)時） 6.56.5影響酶作用的因素（影響酶作用的因素（2 2學(xué)時）學(xué)時） 6.66.6酶催化作用機理（酶催化作用機理（2 2學(xué)時）學(xué)時） 6.76.7結(jié)構(gòu)酶及其作用原理（結(jié)構(gòu)酶及其作用原理（2 2學(xué)時）學(xué)時）第七章第七章 維生素與激素（維生素與激素（6 6學(xué)時）學(xué)時） （周叢照）（周叢照） 7.17.1維生素與輔酶（維生素與輔酶（2 2學(xué)時）學(xué)時） 7.27.2激素概述（激素概述（2 2學(xué)時）

6、學(xué)時） 7.37.3激素作用原理（激素作用原理（2 2學(xué)時）學(xué)時）生物化學(xué)（下）生物化學(xué)（下） 3636學(xué)時學(xué)時第第 八八 章章 代謝總論（代謝總論（2 2學(xué)時）學(xué)時）第第 九九 章章 生物膜和物質(zhì)運輸（生物膜和物質(zhì)運輸（2 2學(xué)時）學(xué)時）第第 十十 章章 糖酵解（糖酵解（6 6學(xué)時）學(xué)時）第十一章第十一章 檸檬酸循環(huán)檸檬酸循環(huán) （6 6學(xué)時）學(xué)時）第十二章第十二章 氧化磷酸化（氧化磷酸化（5 5學(xué)時）學(xué)時）第十三章第十三章 光合作用（光合作用（2 2學(xué)時）學(xué)時）第十四章第十四章 糖原的分解和生物合成糖原的分解和生物合成 （5 5學(xué)時）學(xué)時）第十五章第十五章 脂肪酸代謝（脂肪酸代謝（4 4學(xué)時

7、）學(xué)時）第十六章第十六章 氨基酸的分解代謝（氨基酸的分解代謝（2 2學(xué)時）學(xué)時）第十七章第十七章 核酸代謝（核酸代謝（2 2學(xué)時）學(xué)時） What is Biochemistry? the study of those molecules used and manufactured by living things.Three aspects of biochemistry: 1) Biochemistry is concerned with structural chemistry. It seeks to determine the structures of molecules foun

8、d in living systems in order to understand structure-function relationships. 2) Biochemistry is concerned with chemical change, this is reflected in the study of metabolic pathways 3) Biochemistry is concerned with information which has accumulated through evolution and is preserved in DNA (or somet

9、imes RNA). These nucleic acid sequences code for amino acid sequences, which result in folded proteins. These proteins are often catalysts (enzymes) and some of them are regulated (able to sense the chemical state inside the cell and, in some cases, the outside) Biochemistry through evolutionHow to

10、build a life with molecules? Or The Molecular Design of Life Four transitions through evolution:1, chemicals, micromolecules 2, macrobiomolecules3, energy4, stress responseThe evolution of life required a series of transitions, beginning with the generation of organic molecules that could serve as t

11、he building blocks for complex biomolecules. The next major transition in the evolution of life was the formation of replicating molecules. Evolution Requires Reproduction, Variation, and Selective PressureReplication, coupled with variation and selective pressure, marked the beginning of evolution.

12、 Variation was introduced by a number of means, from simple base substitutions to the duplication of entire genes. RNA appears to have been an early replicating molecule. Furthermore, some RNA molecules possess catalytic activity. However, the range of reactions that RNA is capable of catalyzing is

13、limited. With time, the catalytic activity was transferred to proteins, linear polymers of the chemically versatile amino acids. RNA directed the synthesis of these proteins and still does in modern organisms through the development of a genetic code, which relates base sequence to amino acid sequen

14、ce. Eventually, RNA lost its role as the gene to the chemically similar but more stable nucleic acid DNA. In modern organisms, RNA still serves as the link between DNA and protein.Energy Transformations Are Necessary to Sustain Living SystemsAnother major transition in evolution was the ability to t

15、ransform environmental energy into forms capable of being used by living systems. ATP serves as the cellular energy currency that links energy-yielding reactions with energy-requiring reactions. ATP itself is a product of the oxidation of fuel molecules, such as amino acids and sugars. With the evol

16、ution of membranes hydrophobic barriers that delineate the borders of cells ion gradients were required to prevent osmotic crises. These gradients were formed at the expense of ATP hydrolysis. Later, ion gradients generated by light or the oxidation of fuel molecules were used to synthesize ATP. Cel

17、ls Can Respond to Changes in Their EnvironmentsThe final transition was the evolution of sensing and signaling mechanisms that enabled a cell to respond to changes in its environment. These signaling mechanisms eventually led to cell-cell communication, which allowed the development of more-complex

18、organisms. The record of much of what has occurred since the formation of primitive organisms is written in the genomes of extant organisms. History of Biochemistry(up to 1982)1835 Jons Berzelius chemical catalysis, uses amylase(淀粉酶) as an example.1859 Charles Darwin publishes On the Origin of Speci

19、es. 1860 Louis Pasteur fermation catalyzed by enzymes, essence of yeast. 1865 Gregor Mendel publishes his theory of genetics. 1869 Fredrick Meischer discovers DNA in cell nuclei. 1897 Eduard and Hans Buchner extracts materiel from yeast, conversion of glucose to alcohol. 1914 Fritz Lipmann, the role

20、 of ATP in energy metabolism. 1926 James Sumner, crystalline jack bean (刀豆) urease, is a protein. 1926 Thomas Hunt Morgan writes The Theory of the Gene. 1934 Arnold Beckman developes the first pH meter. 1937 Hans Krebs discovers the citric acid cycle (TCA cycle). 1941 George Beadle & Edward Tatu

21、m, the one-gene, one-enzyme hypothesis. 1944 Oswald Avery, Colin MacLeod, and Maclyn McCarthy DNA is the genetic material. 1950 Edwin Chargaff A=T, G=C (Chargaffs rules). 1952 Linus Pauling and Robert Corey -helix and the -pleated sheet 1953 James Watson and Frances Crick the double helix model of D

22、NA. 1953 Fredrick Sanger the first amino acid sequence of a protein (insulin). 1956 Earl Sutherland isolates cyclic AMP. 1957 Matthew Meselson and Franklin Stahl semiconservative DNA replication. 1960 John Kendrew and Max Pertuz obtain the first 3-D structure of proteins (hemoglobin and myoglobin).

23、1960 Jerald Huritz and Samuel Weiss discover RNA polymerase. 1961 Francois Jacob and Jaques Monod propound the operon model of gene control.1963 Jean-Pierrre Changuex, F. Jacob, and J. Monod Allosteric model for inhibition of enzymes1964 Several groups Acrylamide gel electrophoresis of proteins is d

24、eveloped1965 Marshal Nirenberg, H. Gobind Khorana, and Severo Ochoa complete the elucidation of the genetic code1965 David Phillips 3-D model of first enzyme (lysozyme)1965 Robert Holley determines the structure of a transfer-aaRNA. 1965 Jerome Vinograd discovers superhelical twisting. 1968 Mark Pta

25、shne and Walter Gilbert identify the first repressor genes1969 First synthesis of an enzyme (ribonuclease). 1970 Hamilton Smith discovers restriction endonucleases. 1970 Howard Temin and David Baltimore discover reverse transcriptase.1973 Stanley Cohen and Herbert Boyer prepare recombinant DNA. 1974

26、 Sung-Hou Kim, et al. produce the first X-ray structure of transfer RNA.1977 Cesar Milstein discovers how to produce monoclonal antibodies. 1977 Allan Maxam and Walter Gilbert develop a chemistry for sequencing DNA. 1977 Fredrick Sanger, S. Nicklen and A.R. Coulson develop a chemistry for sequencing

27、 DNA. 1977 Phillip Sharp and Richard Roberts discover introns (intervening sequences). 1982 Amzel LM, McKinney M, Narayanan P, Pedersen PL First x-ray structure of a membrane protein (9). 1980s and early 1990sWonderful time for Molecular BiologyHuman Genome Project (1988-)Central Dogma (Crick F. 195

28、8)Molecular BiologyOmics: Metabolomics Transcriptomics Proteomics Structural Genomics (1998-) GenomicsThe era of Omics:(1998-2003 or later?)Omics = Oh! Mix!(millennium chaos?)What is Structural Genomics?It is an approach aiming at solving 3-D structures of the proteins encoded by an entire genome. K

29、im SH. Nature Structural Biology 1998Original goals of SG:1, to establish a catalogue/library of all folds covering the entire protein universe2, to help the annotation of sequenced genomesInternational Structural Genomics ProjectsUSA and Canada: (1998-) 9 NIH Structural Genomics CentersEU: France (

30、3 centers: Paris-Sud, Marseille, Strasbourg) Germany (Structural Genomics Factory) UK (2 centers) (SPINE:Structural Proteomics in Europe, 2001-)Asia: Japan (RIKEN ) China (Yun-Yu SHI; Zihe RAO ) (2002-) Korea: Israel: Weizmann Structural Proteomics CenterFlow chart:1, Targets selection: Bioinformati

31、cs2, PCR and cloning: Molecular Biology3, Protein production: Biochemistry4, Data collection (X-ray diffraction/NMR)5,Structure determination: Structural Biology6, Function interpretation: General BiologyCore characteristics:High throughput (hundreds to thousands samples)Large-scale(milligram level

32、of protein sample)Multi-discipline integration(from molecular biology to structural biology) high throughput parallelization (平行化平行化) miniaturization (微量化微量化) automation (自動化自動化)cloning robot: 500-1000 clones /3 dayscrystallization robot: 96x3 drops/15 minprotein sample for crystallization trials in

33、 nanoliter volumes (10nl)Hansen CL. et al. PNAS; 2002TagSize (aa)ApplicationSequence or GenBank accession no.His6 or 8affinityHHHHHH(HH)T711 affinityASMTGGQQMGRS15affinityKETAAAKFERQHMDSArg1,5,6affinityRRRRR(R)CBP26affinityKRRWKKNFIAVSAANRFKKISSSGALFLAG4 or 8affinityDYKD or DYKDDDDKStrep 8affinityWS

34、HPQFEKNusA491SET, affinityAccession no. AAN82367 MBP396SET, affinityAccession no. AAC43128 GST220SET, affinityAccession no. AAB59203 ZZ116SET, affinityAccession no. M74186TrxA109SETAccession no. AAC40210 Gb156SETAccession no. 1MPEADsbADsbC208 236SETSETAccession no. P24991Accession no. P21892Zhou CZ

35、& Chen YX. Current Genomics; 2004Different tags will make the difference Different E. coli strains (DE3 series) BL21 (DE3)BL21-pLysSRosettaRosetta-pLysSGoldBL21-Codon-plusTunerStarCo-expression with chaperones(DnaK-DnaJ-GrpE and/or GroEL-GroES) Mainly works for those partially soluble targets Co

36、-purification of chaperones with the target protein Re-aggregation/precipitation after removal of chaperonesEukaryotic expression systems(post-translational modifications)Pichia pastorisSaccharomyces cerevisiaeInsect cellsCell linesVirus-mediatedQuantity and Cost-effectivityDNA shufflingSaturation m

37、utagenesisError-prone PCRTo improve: the solubility, activity and/or stabilityReetz, PNAS; 2004in vitro/directed evolutionProteins samples for crystallization trials:1, pure and homogenous2, stable in solution of low salt concentration, proper pH and reductant (BME, DTT or TCEP)3, at high concentrat

38、ion (normally 10mg/ml)Protein samples for NMR data collection:1, double labeling with 13C and 15N2, no oligomerization or aggregation3, stable for at least one week at RT or 4C4, high expression level (cost-effective)Optimization of xtals:1, about half of the xtals will not diffract at 3 or higher r

39、esolution2, parameters: salt concentration, buffer system, pH, divalent or trivalent irons3, precipitants: PEGs, MPD, salt, alcohol, 4, ligands or substrates/products/analogs5, protein or nucleic acid partners Data collection:X-ray diffraction: in-house X-ray generator synchrotron radiation accelera

40、torNMR spectrometry: 500mHz, 600mHz, 800mHz 900mHz Assignment of function based on structural similarity Reciprocal stimulation and validation: biochemical assays versus structure analyses Structure-directed drug design Function interpretation and application: Assignment of function based on structu

41、ral similarity3-D structureDALI comparisonClosest structuresSimilar molecular function?http:/www.ebi.ac.uk/dali Reciprocal stimulation and validation:biochemical assays vs. structure analyses An example of licT from Bacillus substilisLicT mutant (active)H207D/H269DLicT wt (inactive)Comparison of lic

42、T-wt and licT mutantGraille* and Zhou* et al. JBC 2005 van Tilbeurgh et al. EMBO J. 2001Yang et al. EMBO J. 20021122mRNA1122mRNAPKD=10M KD=1MCATPRD2PRD1RATCATRNA Structure-directed drug design An example of Thy1 from Thermotoga maritima Thy1: thymidylate synthase-complementing protein present in arc

43、haea, prokaryotes, viruses NOT in eukaryotesLesley, SA et al. PNAS; 2002Thy1-FAD-dUMP Thy1-dUMP-HEPES PDB Content Growth (2004/08/01)52,66227,99916,0975,816 5,6992,133 1,031892645010,00020,00030,00040,00050,00060,000targetsclonedexpressedsolublepurifiedcrystallizeddiffraction-qual.diffractionstructu

44、resOutput from International Structural Genomics ConsortiaContribution from crystallographers, 2004/04/132,75583824920005001,0001,5002,0002,5003,000targetsHSQCNMR assignedNMR structuresOutput from International Structural Genomics Consortiium Contribution from NMR spectrometrists, 2004/04/13Future orientations of SG1, Reconstruction of multiprotein complexes (based on interact