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Strukturna biologija, bioinformatika, biologija sistema biologija 21-og veka Danasnja presentacija bice podeljena u tri dela: 1. 2. Pozadina i opsti uvod NMR Rentgenska kristalografija Strukturna biologija – primeri iz moje laboratorije 3. Poseban osvrt na bioinformatiku i biologiju sistema kao interdisciplinarne grane relvantne za kompiuterske nauke Human & other genome sequences Functionally cloned DNA molecules coding for proteins of interest. Bioinformatics Amino acid sequences Protein structure Structural biology Protein function Protein chemistry / yeast 2hybrid screens / proteomics / enzymology / genetics / transgenes / knock-outs / knock-ins / chemical genetics / etc. 3D structures of molecules allow us to understand biological processes at the most basic level. We can ‘see’ which molecules interact, how they interact, how they function, how drugs act. They can help us understand disease at an atomic level. 3D structures can be exploited in development of new drugs. structure-based drug design Strukturna Biologija • Struktura moze ponekad da odkrije funkciju proteina direktno • Struktura moza da racionalizuje eksperimentalnu obzervaciju o selktivitetu i specificnosti enzimaticne reakcije • Struktura moze da postane osnova za rational drug/inhibitor discovery. • Struktura moze da razotkrije dinamicki aspekt proteinskog ponasanja. • Trodimenzionalne topologije polipeptida obezbedjuju podatke za resavanje problema formiranja proteinske strukture -- ‘protein folding problem’. • [Sometimes a 3D structure can be rather uninformative - the ‘structural genomics’ debate.] Experimental modes of molecular structural biology X-ray crystallography NMR spectroscopy Protein crystals Protein solutions (3-50 mgs/ml; ca. 100 ml) (> 0.5 mM; min. volume 0.3ml; 10100 mg) Macromolecular assemblies/particles frozen in vitreous ice 15N,13C-Isotope Electron microscope [Se-methionine labelling] X-ray source/Synchrotron Applicable to proteins of any size (in principle). labelling NMR Spectrometer Applicable to small(ish) proteins (smaller than ca. 30,000 MW) High resolution Medium resolution Cryo-electron microscopy Large particles, typically > 500,000 MW ‘Low’ resolution The sizes of cells and of their component parts Unaided eye 200 mm Light microscope x 10 20 mm x 10 2 mm x 10 200 nm CELLS = 106 mm = 109 nm x 10 20 nm x 10 2 nm x 10 0.2 nm MOLECULES ORGANELLES 1 m = 103 mm Electron microscope ATOMS STA JE NMR? Nuclear Magnetic Resonance (NMR) je mocna spektroskopska tehnika koja pruza informaciju o strukturnim i hemijskim osobinama molekula. NMR je ne-destruktivna metoda za analizu strukture i dinamike molekula. NMR koristi osobine odredjenih atoma kada su izlozeni vrlo jakom magnetnom polju. For biochemists these are mainly 1H, 15N, 13C and 31P. 1H and 31P are highly abundant isotopes whilst 15N and 13C are present at only low levels < 1%. Studies using these nuclei generally require isotopic enrichment by production of the molecule from media that has been enriched in these isotopes. Prof. Kurt Wüthrich Nobel Prize for Chemistry 2002 Typically the magnets used in NMR spectroscopy are 10,000-15,000 times stronger than the earth’s magnetic field. The NMR experiment generally consists of applying short bursts or pulses of energy in the radio frequency (RF) range, typically 40-800 MHz, to the sample. These pulses of RF cause the nuclei to rotate away from their equilibrium position and they start to precess (rotate) around the axis of the magnetic field. The exact frequency at which the nuclei precess is related to both the chemical and physical environment of the atom in the molecule. By using different combinations of RF pulses and delays it is possible to determine how each atom in the molecule interacts with other atoms in the molecule. 110 709 592 15N(ppm) 543 709 592 110 110 115 115 120 120 120 125 125 125 115 732 1H(ppm) 10.0 9.0 8.0 7.0 10.0 9.0 8.0 7.0 10.0 9.0 8.0 7.0 The NMR spectrum is exquisitely sensitive to the conformation of the polypeptide chain, and to the presence of interacting chemical ligands. These and other features of the rich ‘spin physics’ that underlies the NMR phenomenon mean that NMR spectroscopy is a highly versatile tool for the characterisation of: Structure Dynamics Molecular interactions C C a5 a2 C a4 N N a1 N a3 a6 Harris et al. (2004) J. Mol. Biol. C a 3 a5 N a1 C N a4 E676 E667 D709 E664 a2 a4 E700 a6 E699 E652 Harris et al. (2004) J. Mol. Biol. X-ray Crystallography - An experimental technique involving diffraction of X-rays by crystalline material. -X-ray wavelength ~ Å -Based on the diffraction pattern, electron density of the molecule could be reconstructed. (Need intensities and phases) -Model is built in the reconstructed electron-density -Model – the molecular picture – molecular structure from global folds to atomic details -Limited information about the molecule’s dynamic -Depends on obtaining crystals 1. Why X-rays? 2. Why electron density? 3. Why crystals? COMPUTED ELECTRON-DENSITY MAP EYEPIECE LENS magnification n Scattered radiation OBJECTIVE LENS magnification m CRYSTALLOGRAPHER PHASES COMPUTER DETECTOR Scattered radiation OBJECT OBJECT (crystal) VISIBLE LIGHT Enlarged image of object Magnification mn X-RAYS Pregled procesa odredjivanja strukture proteina koriscenjem difrakcije X zraka 1. Proizvodnja izolovanog dovoljno velikog kristala kandidat proteina 2. Postavljanje kristala, prikupljanje i evaluacija preliminarnih difrakcionih podataka 3. Kompletno prikupljanje podataka i procena fazi 4. Izgradnja i rafiniranje proteinskih lanaca 5. Validacija strukture European Synchrotron Radiation Facility (Grenoble, France) Structural characterisation of drug-targets from M.tuberculosis Institute of Structural Molecular Biology Snezana Djordjevic M. tuberculosis • 2-3 million deaths from tuberculosis annually • 1/3 of world population currently infected with the disease • Drug resistance -multidrug-resistant strains -12.6 % M. tuberculosis isolates resistant to at least one drug -2.2 % resistant to both isonazid and rifampin New Drugs -agents that exhibit activity against drug resistant strains -completely sterilize infection -shorten the duration of drug therapy and thus promote drug compliance METRO – 06/03/2007 Mechanism of resistance to Isoniazid -Isoniazid is a prodrug that is oxidized by KatG -KatG is catalase-peroxidase -Mutation of the KatG leads to resistance KatG Prodrug activation Resistance KatG activity is important for virulence ! -Physiological function of the KatG includes protection of the mycobacterium against H2O2 and other ROS produced by the microbe and its host. ? KatG AhpC AhpD AhpD Alkylhydroperoxidase From M. tuberculousis Paul Ortiz de Montellano Dept. of Pharmaceutical Chemistry, UCSF C2; a=186.38 Å, b=117.28 Å, c=88.99 Å, b=113.97° 177 residues/monomer Structure solution: SeMet/MAD 4 wavelengths data collected in Grenoble 1.9 (1.7) Å resolution 2Fo-Fc map AhpD Monomer Topology From structure to function and the catalytic mechanism CXXC a7 a6 Thioredoxins a5 Peroxiredoxins -solvent exposed a3 a8 -pKa ~ 7.1 a4 a2 C a1 N N C Cys130 Cys133 His137 Glu118 Putative substrate binding site Cys133 Novel redox pathway in M. tuberculosis NADH Lpd(ox) DlaT-LpH2 AhpD(ox) AhpC(red) ROOH NAD+ Lpd(red) DlaT-Lp AhpD(red) AhpC(ox) ROH E3 E2 Lpd: Dihydrolipoamide dehydrogenase SucB: Dihydrolipoamide acyltransferase Components of pyruvate dehydrogenase complexes Pyruvate Acetyl-CoA + CO2 NAD+ NADH Molecular Surface A Prototypical Two-Component Signal Transduction System Periplasmic Space P External Stimulus Receptor / input / sensor domain Kinase Core Histidine Kinase (HK) Sensory Protein Response Regulator (RR) Response Chemotaxis P Tar -CH3 B SAM +CH3 W R W A A +ATP P B P Y DosS • Induced by exposure to hypoxia, NO and ethanol. • Structural studies have been initiated with the aim of describing the signalling mechanism that leads to histidine kinase activation. • Histidine kinase domain (HK) undergoes autophosphorylation and can carry out a Mg2+ dependant phosphotransfer reaction onto DosR. • DosS : DosR are a cognate sensor-regulator pair. Identification of domain boundaries Further structural investigation of GAF domains PDE2A_B DosS GAF_A cGMP PDE_1 cGMP PDE_2 anfA cGMP PDE_3 ADEN_CYCL_1 ADEN_CYCL_2 yebR Hypoth. Pro. Nif-regul_1 Nif-regul_2 Nif-regul_3 Nif-regul_4 consensus b1 a2 Secondary Structure: 1MC0 b2 196 3 154 336 46 228 79 271 27 54 68 46 35 21 DVSVLLQEIITEARN-------LSNAEICSVFLLDQ------------NELVAKVFDGGVVDDe----sY DLEATLRAIVHSATS-------LVDARYGAMEVHDRQH---------RVLHFVYEGIDEETVR------R DVTALCHKIFLHIHG-------LISADRYSLFLVCEdss-------ndKFLISRLFDVAEGSTleeasnN SLEVILKKIAATIIS-------FMQVQKCTIFIVDEdcsdsf-ssvfhMECEELEKSSDTLTR------E DLADALSIVLGVMQQ-------HLKMQRGIVTLYDMr----------aETIFIHDSFGLTEEEk-----K DATSLQLKVLRYLQQ-------ETQATHCCLLLVSEd----------nLQLSCKVIGEKVLG-------E GFENILQEMLQSITLkt---geLLGADRTTIFLLDEe----------kQELWSIVAAGEGDRS------L DLEDTLKRVMDEAKE-------LMNADRSTLWLIDRd----------rHELWTKITQDNGST-------K DLNRDFNALMAGETS-------FLATLANTSALLYErlt-------diNWAGFYLLEDDTLVLg----pF LIKATLQKTMEASIH-------QTGAQLGSLFLLDGd----------gRVTESILARGATDQSqk---kN RLEVTLANVVNVLSS-------MLQMRHGMICILDSe-----------GDPDMVATTGWTPEMa-----G RLEVTLANVLGLLQS-------FVQMRHGLVSLFNDd-----------GVPELTVGAGWSEG-------T NTARALAAILEVLHD-------HAFMQYGMVCLFDKe----------rNALFVESLHGIDGERkk--etR DLSKTLREVLNVLSA-------HLETKRVLLSLMQDs-----------GELQLVSAIGLSYEEf-----Q 1 DLEELLQTILEELRQ-------LLGADRVSIYLVDEDK---------RGELVLVASDGLTLPE------L b3 a3 b4 a4 b5 PDE2A_B DosS GAF_A cGMP PDE_1 cGMP PDE_2 anfA cGMP PDE_3 ADEN_CYCL_1 ADEN_CYCL_2 yebR Hypoth. Pro. Nif-regul_1 Nif-regul_2 Nif-regul_3 Nif-regul_4 EIRIPADQ-----GIAGHVATTGQILNIP-DAYAHPl--fYRGVDDSTGFR-----TRNILCFPIKNEnIGHLPKGL-----GVIGLLIEDPKPLRLD-DVSAHP----AS-IGFPPYHPP----MRTFLGVPVRVR-CIRLEWNK-----GIVGHVAAFGEPLNIK-DAYEDPr--fNAEVDQITGYK-----TQSILCMPIKNHrRDANRINY-----MYAQYVKNTMEPLNIP-DVSKDKr---FPWTNENMGNInq-qcIRSLLCTPIKNGkRGIYAVGE-----GITGKVVETGKAIVAR-RLQEHP-----DFLGRTRVSRng-kaKAAFFCVPIMRA-EVSFPLTM-----GRLGQVVEDKQCIQLK-DLTSDD----VQQLQNMLGCE-----LRAMLCVPVISRaEIRIPADK-----GIAGEVATFKQVVNIPfDFYHDPrsifAQKQEKITGYR-----TYTMLALPLLSEqELRVPIGK-----GFAGIVAASGQKLNIPfDLYDHPdsatAKQIDQQNGYR-----TCSLLCMPVFNGdQGKIACVRipvgrGVCGTAVARNQVQRIE-DVHVFD-------GHIACDAA-----SNSEIVLPLVVK-IVGQVLDK-----GLAGWVRENKRTGLIN-DTTKDY----RWLKLPDEPYQ-----ALSALGVPIVWG-QIRAHVPQ-----KAIDQIVATQMPLVVQ-DVTADP-----LFAGHEDLFGppeeaTVSFIGVPIKAD-DERYRTCVp---qKAIHEIVATGRSLMVE-NVAAEt---aFSAADREVLGAsd-siPVAFIGVPIRVD-HVRYRMGE-----GVIGAVMSQRQALVLP-RISDDQ-----RFLDRLNIYDy----SLPLIGVPIPGAdSGRYRVGE-----GITGKIFQTETPIVVR-DLAQEP-----LFLARTSPRQsqdgeVISFVGVPIKAA-- consensus GVRFPLDE-----GLVGRVAETGRPLVIP-DVEADP----FFFLDLLQRYQL----IRSFLAVPLVAG-- Secondary Structure|1MC0 b6 a5 PDE2A_B DosS GAF_A cGMP PDE_1 cGMP PDE_2 anfA cGMP PDE_3 ADEN_CYCL_1 ADEN_CYCL_2 yebR Hypoth. Pro. Nif-regul_1 Nif-regul_2 Nif-regul_3 Nif-regul_4 -QEVIGVAELVNK-------------------INGPWFSKFDEDLATAFSIYCGISIAHSLLYKKVN -DESFGTLYLTDK-------------------TNGQPFSDDDEvlvqalaaaagiavanarlyqqak -EEVVGVAQAINKk-----------------sGNGGTFTEKDEKDFAAYLAFCGIVLHNAQLYETSL kNKVIGVCQLVNKmee--------------ttGKVKAFNRNDEQFLEAFVIFCGLGIQNTQMYEAVE -QKVLGTIAAERV-------------------YMNPRLLKQDVELLTMIATMIAPLVELYLIENIER tDQVVALACAFNK-------------------LGGDFFTDEDERAIQHCFHYTGTVLTSTLAFQKEQ -GRLVAVVQLLNKlkpyspp-----dallaerIDNQGFTSADEQLFQEFAPSIRLILESSRSFYIAT -QELIGVTQLVNKkktgefppynpetwpiapeCFQASFDRNDEEFMEAFNIQAGVALQNAQLFATVK -NQIIGVLDIDST--------------------VFGRFTDEDEQGLRQLVAQLEKVLATTDYKKFFA -DELLGILTLMHS--------------------QVNHFTPACATAMEKTAELIALVLNNARIQTKHK -HHVMGTLSIDRIw-----------------dGTARFRFDEDVRFLTMVANLVGQTVRLHKLVASDR -STVVGTLTIDRIp------------------EGSSSLLEYDARLLAMVANVIGQTIKLHRLFAGDR -NQPAGVLVAQPM-------------------ALHEDRLAASTRFLEMVANLISQPLRSATPPESLP -REMLGVLCVFRDg------------------QSPSRSVDHEVRLLTMVANLIGQTVRLYRSVAAER consensus -GELLGVLALHRK-------------------DSPRPFTEEEEELLQALANQLAIALALAQLYEELR 345 150 314 503 196 375 249 441 179 202 220 198 186 180 SAMt99 : to detect remote structural homologues of this protein. From the 11149 sequence homologies identified, 24 had a known structure but none of those identified produced significant global alignment. Local alignments covered either the C or N terminal regions. No alignment was found that covered both putative GAF domains. 1 structural homologue was identified for DosS GAF A domain : 1MC0 UV-Visible Characterisation of GAF A Haem Absorption Haemoglobin Absorptionspectra spectra ofof Haemoglobin Absorption spectraof of DosS DosS 63-210 Absorption spectra GAF A A 0.1 A. Oxy-ferrous (dashed line) B. Ferric (solid line) C. Ferrous (dotted line) D. Ferrous-CO (solid line) A. Ferric haemoglobin (solid line) B. Oxy-ferrous haemoglobin (dashed line) C. Ferrous haemoglobin (dotted line) D. Ferrous-CO haemoglobin (solid line) E. Ferrous-NO haemoglobin (solid line) CO / NO / O2 E. Ferrous-NO (solid line) Fe2+ A 0.005 His 500 550 600 650 700 Wavelength (nm) 550 600 Wavelength (nm) 650 Visible/UV spectrum of the DosS GAF A (63-210) histidine to alanine mutants 0.12 H73A H89A H93A 0.10 H97A H113A 0.08 H149A DosS 63-210 0.06 0.04 0.02 40 2 41 3 42 4 43 5 44 6 45 7 46 8 47 9 49 0 50 1 51 2 52 3 53 4 54 5 55 6 56 7 57 8 58 9 60 0 0.00 Wavelength (nm) Absorbance H139A The Model of Signalling O2 Fe2+ A OFF B DosR NO Fe2+ P A B ON P DosR GAF B - NMR 1H, 15N labeled DosS GAF B HSQC NMR experiments: HNCO, HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH, HA(CA)NH and HA(CACO)NH were obtained at 1H frequency of 500MHz on a 0.6mM [1H, 13C, 15N]-labelled DosS 231-379, pH6, 20mM phosphate, 100mM NaCl. GAF B - NMR PROBLEMS: - 48 residues are still to be assigned - 21 expected cross-peaks are missing from the spectrum. Sekharan MR, Rajagopal et al. 2005. Backbone 1H, 13C, and 15N resonance assignment of the 46 kDa dimeric GAF A domain of phosphodiesterase 5 J Biomol NMR. 33(1):75 - Some of the cross-peaks do not form one peak but multiple peaks. Predicted secondary structure for DosS GAF 2 using PSIPRED. - High content of Val, Leu and Ala residues in the sequence. Signalling mechanism N C STRUCTURAL GENOMICS CENTRES IN NORTH AMERICA, UK, FRANCE, JAPAN • OXFORD STRUCTURAL GENOMICS • Announced in 2003, with operations commencing in July 2004 for an initial three-year period, this initiative received funding from Canadian, Swedish and British sponsors from both the public and private sectors. For the second phase, July 2007, over £49 million is being made from public funding agencies in Canada, Sweden and Ontario, charitable foundations in the UK and Sweden, GlaxoSmithKline plc, Novartis and Merck. Laboratories at the University of Oxford , University of Toronto and Karolinska Institutet, Stockholm. BIOINFORMATIKA U toku poslednjih nekoliko dekada, napredak u molekularnoj biologiji, zajedno sa progresom u genetskoj tehnologiji doveo je do eksplozije u kolicini informacija stvorenih u naucnoj zajednici. Pojava te mase informacija proizvela je potrebu i zahtev za kompiuterizovanim bankama podataka (databases) da bi se cuvali, organizovali i katalogovali podaci. Pritom neophodno je bilo razviti sredstva (tools) za pregled, vizualizaciju i analizu tih podataka. Computational biology (sam proces analize i interpretacije podataka) • Razvoj i primena alatki (tools) koji omogucavaju pristup, upotrebu i organizaciju raznih informacija • Razvoj novih algoritma i statistike sa kojima se mogu proceniti relazije medju komponentama u velikoj grupi podataka. Na primer metode za lociranje gene u okviru sekvence, predvidjanje strukture proteina/funkcije, i grupisanje proteinskih sekvenci u familije povezanih (related) slicnih sekvenci. “Organizmi funkcionisu kao integrisani sistemi – nasa cula, nasi misici, nas metabolizam i nas um rade zajedno u povezanoj celini. Biolozi su tradicionalno proucavali organizme deo po deo i uzivali u modernoj moci da proucavaju molekul po molekul, gen po gen. ISM je posvecen novoj nauci, kriticnoj nauci buducnosti kojoj je za cilj da razume integraciju delova koji sacinjavaju bioloski system.” David Baltimore (Nobel Laureate) President, Cal. Institute of Tech., Pasadena Systems biology requires: -Integration of biology, technology, computation medicine -a strong cross-disciplinary team of researchers. -Institutes include scientists trained in biology, physics, chemistry, engineering, computing, mathematics, medicine, immunology, biochemistry, and genetics. -They all speak the language of biology assembled into a multiplicity of teams that are attacking focused and important problems of systems biology. Health Care in the 21st Century: • Predictive (genetic makeup, protein markers) • Preventive (probability of disease and response to treatment) • Personalized (customized therapeutic drugs) http://csbi.mit.edu:8080/infoglueDeliverWorking/ The MIT CSBI links biologists, computer scientists and engineers in a multi-disciplinary approach to the systematic analysis of complex biological phenomena. http://www.sbml.org/Main_Page The Systems Biology Markup Language (SBML) is a computer-readable format for representing models of biochemical reaction networks in software. It's applicable to models of metabolism, cell-signaling, and many others. SBML has been evolving since mid-2000 thanks to an international community of software developers and users. This website is the portal for the global SBML development effort; here you can find information about all aspects of SBML. Manchester Centre for Integrative Systems Biology (MCISB) • Molecular Biology / Biochemistry / Biophysics), mathematical and computational (Modelling / Data Integration / Text Mining • Development and exploitation of methods for the quantitative measurement of kinetic and binding constants on a genome-wide scale • Combined approaches will lead to computer models of parts of living cells. Some of these 'silicon cells' are already available for in silico experimentation, through the Biomodels and JWS databases. VIRTUELNA CELIJA Acknowledgements: My Group: Sunita Sardiwal Syeed Hussain Shreenal Patel Mark Jeeves Christine Nunn NMR: Paul Driscoll Richard Harris Collaborators at RVC: Neil Stoker Sharon Kendall Farahnaz Moahedzadeh Stuart Rison UV-VIS Spectra Peter Rich Doug Marshall PHOSPHORYLATION Studies: Irina Tsaneva EM: Helen Saibil Nadav Elad ITC/CD: John Ladbury Paul Leonard