1、蛋白質結構與功能IIXiao-Dong Su蘇曉東Biodynamic OPtical Imaging CenterSchool of Life Sciences, Peking UniversityDecember 04, 2013 上海生命科學?statistics/p=generalinformation/pdbindex.htmlPDB ID: #XYZ.pdb常用圖像程序（顯示蛋白質及其復合物的三維結構）：PyMOL (MacPymol); Rasmol;Molmol; HEADERSTRUCTURAL PROTEIN09-JUN-011JCETITLEMREB FROM THERM

2、OTOGA MARITIMACOMPND COMPND COMPND COMPND SOURCE SOURCE SOURCE SOURCE SOURCE SOURCE SOURCE SOURCE SOURCE KEYWDS KEYWDS EXPDTA AUTHOR REVDAT REVDAT REVDAT JRNL JRNL JRNL JRNL JRNL JRNLMOL_ID: 1;2 MOLECULE: ROD SHAPE-DETERMINING PROTEIN MREB;3 CHAIN: A;4 ENGINEERED: YESMOL_ID: 1;2 ORGANISM_SCIENTIFIC:

3、 THERMOTOGA MARITIMA;3 ORGANISM_TAXID: 2336;4 GENE: TM0588;5 EXPRESSION_SYSTEM: ESCHERICHIA COLI;6 EXPRESSION_SYSTEM_TAXID: 562;7 EXPRESSION_SYSTEM_STRAIN: C41;8 EXPRESSION_SYSTEM_VECTOR_TYPE: PLASMID;9 EXPRESSION_SYSTEM_PLASMID: PHIS17ROD-SHAPE DETERMINING, MREB, MBL, ACTIN, HSP-70, FTSZ, 2 STRUCTU

4、RAL PROTEINX-RAY DIFFRACTIONF.VAN DEN ENT,L.A.AMOS,J.LOWE324-FEB-09 1JCE225-DEC-02 1JCE119-SEP-01 1JCE110VERSN REMARKAUTHF.VAN DEN ENT,L.A.AMOS,J.LOWETITLPROKARYOTIC ORIGIN OF THE ACTIN CYTOSKELETON.REFNATURE REFNPMID11544518V. 413ISSN 0028-083639 2001DOI10.1038/35092500REMARK1REMARK2REMARK2 RESOLUT

5、ION.2.10 ANGSTROMS.ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM ATOM1234567891011121314151617181920212223242526272829303132NLYS A CA LYS A CLYS A OLYS A44444444455555-2.731 31.328 31.514 1.0

6、0 53.53-1.592 31.179 30.561 1.00 54.77-0.913 29.819 30.745 1.00 52.59-0.412 29.504 31.823 1.00 54.06N C C O C C C C N NC C OC C O ON C C O CC C C N C C O N C CCB CG CD CE NZ NLYS A LYS A LYS A LYS A LYS A ASP A-0.5810.5781.5610.9600.725-0.90832.30732.31233.43634.81035.05829.01630.78229.79930.10729.8

7、4528.39629.6881.00 57.811.00 64.161.00 66.471.00 68.091.00 66.191.00 48.73CA ASP A C ASP A O ASP A-0.305 27.689 29.734 1.00 47.250.763 27.533 28.658 1.00 42.940.567 27.938 27.509 1.00 40.00-1.383 26.622 29.543 1.00 56.75-2.341 26.541 30.714 1.00 63.70-3.347 25.806 30.603 1.00 66.10-2.087 27.204 31.7

8、44 1.00 68.61CB ASP ACG ASP A OD1 ASP A OD2 ASP A555N ILE A CA ILE A CILE AO ILE ACB ILE A CG1 ILE A CG2 ILE A CD1 ILE A NGLY A CA GLY A6666666677771.887 26.930 29.028 1.00 40.052.983 26.749 28.086 1.00 35.693.553 25.339 28.054 1.00 33.073.591 24.641 29.068 1.00 31.964.154 27.706 28.404 1.00 34.994.

9、7263.6835.93827.38229.14628.20929.78728.35830.1671.00 38.851.00 37.521.00 43.513.995 24.935 26.867 1.00 31.484.617 23.641 26.694 1.00 26.196.094 23.944 26.530 1.00 27.936.459 24.908 25.846 1.00 23.946.952 23.141 27.151 1.00 24.168.384 23.386 27.061 1.00 21.969.224 22.177 26.671 1.00 23.18C O NGLY A

10、GLY AILE A8CA ILE A8 CILE A8The theoretical physicist Richard Feynman once said：“the most important thing about matter is that its built of atoms”“ everything that living things do canbe understood in terms of the jigglings and wigglings of atoms”DNA雙螺旋折疊到的層次結構9es, being supramolecular complexes of

11、mainly nucleic acids and proteins, allow us to analyzemany molecular events in cells in a unique manner.今天的主要內容： 蛋白內的機理及結構生物學研究； DNA 蛋白質的結合及相互作用； 蛋白質結構的研究方法- 過去、現在與未來； 蛋白質的動態(tài)結構及研究方法（單物物理）。生今天的主要內容： 蛋白內的機理及結構生物學研究； DNA 蛋白質的結合及相互作用； 蛋白質結構的研究方法- 過去、現在與未來； 蛋白質的動態(tài)結構及研究方法（單物物理）。生How are proteins made?The

12、central dogma 中心法則Flow of information in gene expressionCodonmRNAAGCTCCTTGAAGTyrValArgTyrArgaa1aa11 aa12aa10Valaa7aa13aa14aa2aa9aa3Proteinaa8How are proteins made?From Anders Liljas1955: Cricks adaptor hypothesis,“It is therefore a natural hypothesis that the amino acid is carried to the template by

13、 anadaptor moleculeand the adaptor is the part which actually fits onto the RNA. In the simplest form one would requiretwenty adaptors,one for each amino acid“Crick suggested that the adaptor could be an RNA-molecule.From Anders LiljasHow is transla-on performed?Cricks adaptor hypothesis:mRNABase pa

14、iringtRNAAmino acidadaptorGrowing polypep7deFrom Anders LiljasThe adaptor tRNAThe hypothesis about the adaptor coincided with the purification of. (1957).tRNA by HoaglandGAmino- acidCrick was surprized when he learnt that the tRNA-molecule was 70-80 nucleotides in length.AC CThe structuren of tRNA p

15、roposed by Watson 1964From Anders LiljasThe tRNA history1957tRNAisolated (Hoagland)1965The first tRNAsequence1974The structure of tRNAPheFrom Anders LiljastRNA:the substrate in translation.RNA has the shape of anL.75Å between anticodon and the acceptor end.The ribosome must be constructed to fi

16、tFrom Anders LiljasTranslation is performed on ribosomesProteinFrom Anders LiljasRibosomeComposi-on of ribosomesSourceRibosomesSmall RNA30SsubunitProteinsLarge RNA50SsubunitProteins33Bacteria70S16S2123S,5SArchaea70S30S16S2850S23S,5S40Mitochondriayeast mammals74S55S50S37S28S30S15S12S9S31297754S38S40S

17、21S16S12S464856TrypanosomesEukarya80S40S18S3260S28S,5S,5.8S46From Anders Liljas12S RNA from bovine mitochondria16S RNA from T. thermophilusFrom Anders LiljasRela7onship between ribosomal proteinsEukaryaArchaeaE:11(4,7)AE:31(13,18)A:1(0,1)BAE:36(15,21)BE:0BA:0B:21(8,13)BacteriaThe transla7on system i

18、s the most conserved molecular system.Watson (1964)concerning the transla7on system:“Unfortunately, we cannot accurately describe at the chemical level how a molecule func7ons unless we know rst its structure.”FUNCTION IS STRUCTURE !From Anders LiljasNobel prize in chemistry 2009Venkatraman Ramakris

19、hnan MRC Laboratory of Molecular Biology, Cambridge, UKThomas A. Steitz Yale University, New Haven, CT, USAAda E. Yonath Weizmann Ins7tute of Science,Rehovot, Israel“for studies of the structure and func5on of the ribosome”The ribosome translates the mRNAsequence into protein sequenceThe structure o

20、f the large and smallribosomal subuSchematic surface rit23S RNA: orange5S RNA: yellow proteins: blue Active site: greenBan, Nissen, Hansen, MooNissen, Hansen, Ban, MooHow does translation work?The mRNA/ tRNA channelmRNAFrom Anders Liljas31The peptide exit channelMicrographStained samples Preferred o

21、rientations Reconstruction “by eye”.Cryo-EM imageflash freezing, vitreous ice Random orientations Imagereconstruction.32The ribosome is a large RNA-protein enzymecomposed of two subunitsproteinsRNALarge Subunit, 50S31(L1, L2, L3)2(23S, 5S)Small subunit, 30S21(S1, S2, S3)1(16S)33Ribosomal proteinsMan

22、y are extremely extended with long tails or well separated domains.This may explain the difficultyto crystallize many of them.From Anders Liljas34Cryo-EM studies reveal low resolution images that may beinterpreted by independently solved structuresCryo-EM map 20 Å resolutioncrystal structureato

23、mic mEF-Tu. (1995)35Stark. (1997)NissenThe Elongation Cycle studied by Cryo-EMEPAR 1 5 2 4 3 36How do 100 RNA helices in 6 RNA domainspack together and form a compact structure?37Proteins provide positive chargeto stabilize the RNARNA stripped awayBlue: positively charged protein regions Red: negati

24、vely charged protein regions38DNA and RNA polymerases areproteins.What forms the peptidyltransferase centre on the 50Ssubunit -RNA or protein?3940The Ribosome is a Ribozyme!RNA makes protein.41The large ribosomal subunit contains a100 Å long tunnel for product release - the polypeptide exit tun

25、nel.Non-stick surface - “teflon” mixed surfaceof hydrophobic and polar patches42今天的主要內容： 蛋白內的機理及結構生物學研究； DNA 蛋白質的結合及相互作用； 蛋白質結構的研究方法- 過去、現在與未來； 蛋白質的動態(tài)結構及研究方法（單物物理）。生DNA Binding Mechanism Revealed by High Resolution Crystal Structure of Arabidopsis thaliana WRKY1 ProteinMing-Rui Duan, Jie Nan, Yu-He

26、Liang, Peng Mao, Lu Lu, Luhua Lai, Yi Li, and Xiao-Dong Su, “DNA Binding Mechanism Revealed by High Resolution Crystal Structure of Arabidopsis thaliana WRKY1 Protein” Nucleic acid research (2007) 35(4):1145-54Crystal structure of human CLOCK and BMAL1 bHLH domains and their tetramer formNon-Specifi

27、c interactions Target Search on DNA- Combined 1D and 3D searchBerg, Winter, von Hippel, Biochem. (1981)今天的主要內容： 蛋白內的機理及結構生物學研究； DNA 蛋白質的結合及相互作用； 蛋白質結構的研究方法- 過去、現在與未來； 蛋白質的動態(tài)結構及研究方法（單物物理）。生:結構生物學研究的主要技術 顯微方法（衍射或散射）： X射線晶體學（X-ray crystallography）優(yōu)點：方法很成熟，可高通量化，分辨率高，可快速測定結構； 缺點：需要可衍射單晶，有相位問題 。 電子顯微學（el

28、ectron microscopy：EM），Cryo EM優(yōu)點：不需大單晶，無相位問題，可觀察原位及單粒子結構；缺點：分辨率低，儀器昂貴，操作復雜；與高分辨X光結構結合，前景可觀。 小角散射方法：X射線（SAXS），可見光。 X射線自由電子激光（XFEL）：前途無量！ 波譜方法： 核磁共振（NMR）優(yōu)點：溶液構象，無需結晶，無相位問題，可得到動力學信息；缺點：方法尚在發(fā)展中，目前仍限于較小量蛋白質結構測定。 其他譜學方法：例如各種光譜學方法（可見；紫外；熒光；紅外；園二色；RAMAN等等）；EPR；EXAFS 質譜方法：Crystallization& Diffractionpatte

29、rnThe use of recombinant proteins 重組蛋白76已經宣布2014年為國際晶體學年 (IYCr 2014)77結構生物學的意義 結構生物學在“后組”生物學中的地位：在蛋白質一級結構序列信息基本已知后，三維原子 結構已成為下一步也是最后一步為全面深入理解生命奧秘所必需的結構信息。因此，結構組學應運而生。 蛋白結構以及大相互作用是揭示生物功能及研究人類疾病機理的基本前提。大結構也是合理設計和蛋白質工程等應用研究的重要基礎。結構組學研究的意義 由結構探索未知功能；（如Pfam）的代表性出每一種蛋白三維結構（最好是高分辨率結構），得到產物的最終信息； 搜尋未知折疊類型（F

30、old）空間，探索蛋白質及折疊問題。Functional proteins are organized by domainsSGs first task: Sequence the domains (or families)From Science. 2003 Apr 18;300(5618):445-52Some examples of domainstructuresModerntechniques allow us to see what could not be seen before.各種光源的分辨極限光學顯微鏡分辨率低于 0.2 µmX-ray分析范圍低于 0.5

31、97;電鏡的分辨率低于 1.9 Å<-肉眼-光鏡-電鏡-X射線晶體學-Technical devepments that greatlypromote Protein CrystallographyBesides crystallogaphic methods: X-ray sources and computers; Protein purification and crystallization. 1950s-60s-70s: MIR/SIR and Computer; 1980s-90s: Recombinant proteins and SR/MAD; 1990s-00

32、s: MAD/SAD and advanced computers; 2000s-10s: SG (Structural Genomics) 2010s-20s: XFEL: X-ray自由電子激光85 FreeFrom Joachim Stohr, SSRL Deputy DirectorWe are hereeach pulse:1012 photons< 100 fs coherentelectron lasersGrowth of X-Ray Brightness and Magnetic Storage Density數據分析：1998 D. Sayre et al.2005D

33、. Shapiro 2008D. Sayre.Gaffney, K.J. & Chapman, H.N. 2007Science 316 14441971R.W. Gerchberg & W.O. Saxton1952 D. Sayre1968D.J. De Rosier &A. Klug單的X射線衍射分析實驗裝置：CF光源：LCLS（美國）Serial femtosecond crystallography of in vivo TbCatB crystals.(a) Diffraction pattern of a TbCatB in vivo crystal re

34、corded from a single shot of 70 fs FEL X-rays. (b) Sum of 988 single-shot FEL diffraction patterns from TbCatB crystals in different orientations. The lower panel of the detector was shifted to achieve higher resolution(Online Methods). At the edge of the detector, aum resolutionof 7.5 Å was ob

35、tained. (c) Precession-style image of the 001 zone for TbCatB, obtained by merging SFX data from 328 in vivo crystal patterns indexed with unit cell constraints. Intensities of integrated生物大結構與功能的關系：功能=運動著的結構生命是由蛋白復合物納米運轉著 Some say: Function is structure! Others say: Structure is function! I say: Fu

36、nctions are structures in motion!Proteins in ActionView of conformational change:利用已知酶底物、酶中間產物、酶產物等復合物晶體結構可以模擬酶反應機理ØØAK催化的磷酸轉移反應循環(huán)中，底物的結合和離開誘導的酶構象變化的示意圖（引自Berry., 1994)The Nobel Prize in Chemistry 2013Martin Karplus, Michael Levitt, Arieh WarshelMartin KarplusMichael LevittArieh WarshelThe

37、 Nobel Prize in Chemistry 2013 was awarded jointly to Martin Karplus, Michael Levitt and Arieh Warshel "forthe development of multiscale m chemical systems".s for complexRapid Developing Biomedical Technologies include: 單技術（Single molecule technology）； 超高分辨率動態(tài)光學成像 (Super-High resolutiondyn

38、amic optical imaging)； 新一代高通量深度技術(NGS Deep Sequencing)99-Modified from Sunney XieNonspecific Binding and Diffusion along DNAIn Vitro Flow-Stretching AssayPaul C. Blainey, et. al. PNAS, 2006N (t) = A exp(- t )t10 mAverage binding 7me = tStandard error = t /sqrt(N)101ko= 1/ tNonspecific Binding and Di

39、ffusion along DNAIn Vitro Flow-Stretching AssayBlainey., PNAS 103, 5752 (2006).Flow àà-Modified from Sunney10X2 ieTranscriptional control in biology103Anders Liljas. (2009) World Scientific, Singapo104105106107108109Wikipediahp:/202.204.115.67/jpkch/jpkch/2008/wswx/Allosteric Regulation (P

40、rotein)- The Hemoglobin CaseOxygen-hemoglobin dissociation curveallosteryThetermcomesGreek wordsfromtheallos (),and “other” stereos () solid(object),inreferencetothefactthattheregulatoryallosteric physicallysiteofan isproteindistinct site.fromitsactiveJacquesLucienMonod(9 February 1910 31 May1976) w

41、as a French biologist who was awarded in 1965 the Nobel Prize in Physiology or Medicine111Modified from Sunney XieYFP labeledlac repressorDNAThe Lac Operon112cell outer membranecell inner membranelac ylac zOffOnOffOnFine Tuning微調113Allostery through DNA?Question: Can a protein bound on DNA affect an

42、other proteins binding affinearby without direct protein-protein interactions?114115116ko of LacR is modulated by binding of T7 RNApas a func-on of their separa-on117Amplitude of the oscilla-on could be adjusted by the proper-es of the linker DNAand “Bubble”1.6If DNA isedIf DNA has mismatched bases1

43、.41.21.00.80.60.405101520253035404550Separation, L (bp)koff(saturating T7 RNAp) / koff(no T7 RNAp)DNA allostery is an intrinsic property of DNAbindingbindersDNA hairpin also has allosteric effect on proteinDNA Allostery exists between two straightMD simula-on to show that the DNA allosteryis mainlyd

44、ue to the conforma-onal change and transducing in the major grooves120Correlation coefficientDistributionTime-correlationATG CF(i0)4.69121.99801315ATGCrystal and Diffraction dataCrystal and Diffraction dataStructure of GRDBD-9GC3Structure alignment with 1R4R(GRDBD-normalGRE)1R4RGRDBD-9GC3 GRDBD-4GC3

45、 GRDBD-9GC3Preliminary results for transcrip-onal eectsCaused by DNA allostery.126Physiological relevance of DNA allosteryDNA Allostery in E. coli Cell127Physiological relevance of DNA allosteryTF128Allostery in Nucleosomal DNADNA Allostery near a NucleosomeLGR between two nucleosomesLL129大家！與晶體衍射及晶

46、體結構有關的部分諾貝爾獎獲得者名單（未包括探測器方面的物理獎項）年份學科獲獎者獲獎原因1901物理德國倫琴Wilhelm Conral RontgenX射線的發(fā)現1914物理德國勞埃Max von Laue晶體的X射線衍射1915物理英國英國亨利.布拉格Henry Bragg勞倫斯.布拉格Lawrence Bragg.晶體結構的X射線分析及解析1917物理英國巴克拉Charles Glover Barkla元素的特征X射線1924物理瑞典卡爾.西格班Karl Manne Georg SiegbahnX射線光譜學1927物理美國康普頓Arthur Holly Compton發(fā)現康普頓效應（散射）

47、1936化學荷蘭德拜Petrus(Peter) J W Debye研究偶極矩和X射線衍射法1937物理美國英國戴維森Clinton Joseph Davisson湯姆孫George Paget Thomson發(fā)現電子衍射1954化學美國鮑林Linus Carl Pauling化學鍵的本質1962化學英國英國肯德魯John Charles Kendrew帕魯茲Max Ferdinand Perutz蛋白質（肌紅蛋白/血紅蛋白）的晶體結構測定1962生理醫(yī)學英國美國英國克里克Francis H.C. Crick沃森James Watson威爾金斯Maurice H.F. WilkinsDNA的雙螺

48、旋結構1964化學英國霍奇金Dorothy Crowfoot Hodgkin青霉素、B12晶體結構測定1976化學美國利普斯科姆William Nunn Lipscomb Jr.硼烷、碳硼烷的結構1979生理醫(yī)學美國英國科馬克Allan MacLeod Cormack豪斯菲爾德Godfrey Newbold Hounsfield用電子計算機操縱的X 射線斷層掃描儀1985化學美國美國霍普特曼Herbert Hauptman卡爾勒Jerome Karle直接法解析晶體結構1986物理德國德國131魯斯卡E. Ruska賓尼希G. Binnig透射電子顯微鏡掃描隧道顯微鏡表二、與生物大分子結構特別

49、是蛋白質研究相關的部分諾貝爾獎獲得者名單年份學科獲獎者獲獎原因1901物理德國倫琴Wilhelm Conral RontgenX射線的發(fā)現1914物理德國勞埃Max von Laue晶體的X射線衍射1915物理英國英國亨利.布拉格Henry Bragg勞倫斯.布拉格Lawrence Bragg晶體結構的X射線分析及解析1926化學瑞典斯維德伯格Theodor（The）Svedberg超速離心分析蛋白質結構1946化學美國美國美國薩姆納James B. Sumner諾思羅普John H. Northrop斯坦利 Wendell Meredith Stanley發(fā)現結晶蛋白酶，制備活性狀態(tài)的酶和病

50、毒蛋白質1948化學瑞典梯塞留斯Arne Wilhelm Kaurin Tiselius發(fā)明電泳方法分析蛋白質1954化學美國鮑林 Linus Carl Pauling化學鍵的本質及蛋白質結構1958化學英國桑格 Frederick Sanger發(fā)明蛋白質測序方法1962化學英國英國肯德魯 John Charles Kendrew帕魯茲 Max Ferdinand Perutz蛋白質（肌紅蛋白/血紅蛋白） 的晶體結構測定1962生理醫(yī)學英國美國英國克里克 Francis H.C. Crick沃森 James Watson威爾金斯 Maurice H.F. WilkinsDNA的雙螺旋結構的提出1964化學英國霍奇金 Dorothy Crowfoot Hodgkin